The film starts with the four humanoid teenage mutant ninja turtles Leonardo (Pete Plozek), Donatello (Jeremy Howard), Raphael (Alan Ritchson), and Michelangelo (Noel Fisher) sliding into Madison Square Garden to catch a basketball game by hiding in the jumbotron. Outside, Vern (Will Arnett) is enjoying his new celebrity status as "The Falcon" since the Turtles let him take credit for bringing Shredder (Brian Tee) down a year earlier in the previous film. Mikey and Raph decide to mess with Vern by throwing spitballs at him.
Meanwhile, April (Megan Fox) is investigating scientist Baxter Stockman (Tyler Perry), thinking he is up to something suspicious. She sits down with him, pretending to be a big fan of his work, and she leaves once she gets her information. April tells Donnie that she found out Stockman is working for Shredder and is trying to set him loose since Shredder is being transported to another prison soon. The Turtles have to flee when Mikey drops a slice of pizza out of the jumbotron, causing one of the players to slip.
Prison guard Casey Jones (Stephen Amell) is set to transport Shredder, along with two other crooks, Bebop (Gary Anthony Williams) and Rocksteady (Sheamus). The two of them are fans of the villain. As they are being taken away, a team of Foot Ninjas pursue the prison van and blow up the security vans in front of them. The Turtles drive by in their own van and shoot manhole covers at the ninjas. Mikey brings out some huge nunchucks and gets a few hits before accidentally destroying them under the bridge. A ninja makes an opening for Shredder to break out of the van, while Bebop and Rocksteady are given a chance to take over the van. We see Stockman in his lab getting ready to teleport Shredder with Karai (Brittany Ishibashi) by his side. Shredder tries to board a chopper overhead while the Turtles try to catch him with a net. Shredder jumps and is then thrown into a portal, leaving the Turtles baffled.
Shredder is brought to Dimension X where he encounters the alien creature known as Krang (Brad Garrett). Krang is a slimy pink brain-squid-thing that uses a giant robot to move around. He tells Shredder to help him gather the pieces to a portal to bring him to Earth where he can unleash a device called the Technodrome so that he may rule over Earth. Stockman already has one piece, and the other two are located at the Museum of Natural History and a rainforest in Brazil. Shredder agrees on the condition that Krang help him eliminate the Turtles. Krang accepts the proposal and sends Shredder back to Earth.
The Turtles sneak back to the sewers. Mikey sees the Halloween parade happening, which he wants to join in since he knows he'll fit in. His brothers make him come back. They slide down and interrupt Splinter (Tony Shaloub) during his meditation.
Chief Rebecca Vincent (Laura Linney) chastises Casey for the escape of the three convicts. Casey tries to defend himself by stating what he saw, but it sounds too farfetched to be believable. Vincent orders him to stay off the case. However, Casey finds the police file on Bebop and Rocksteady and finds out their favorite bar to hang out at, knowing they would be dumb enough to go back there.
Sure enough, Bebop and Rocksteady are at the bar plotting to start their own criminal organization since they think Shredder is out of the picture. He appears before them in his suit and demands that they be his errand boys, which they accept. Casey later arrives at the bar and asks the bartender where to find the crooks, or else he'll keep trashing the bar. The bartender gives him a GPS to use to track them from a signal in the phones he gave them (which were supposed to be untraceable).
Shredder brings the two to Stockman's lab at the TCRI building. Stockman has developed a formula using a special brand of mutagen that can create powerful beings. Shredder takes two darts and shoots them at Bebop and Rocksteady. The two begin to mutate into bigger and stronger creatures. Bebop becomes a warthog and Rocksteady turns into a rhino. Meanwhile, April has snuck into the lab and she steals the mutagen sample.
She runs out into an alley where she is cornered by Foot Ninjas. April thinks that the Turtles will show up to rescue her, but instead, it's Casey (who tracked Bebop and Rocksteady's signal to the building) who shows up with a hockey mask and stick, hitting pucks at the ninjas to take them out. Police arrive and take the mutagen sample (after a ninja took it from April). The Turtles then arrive after the fight is done. They bring Casey to their lair where they trick him into trying to attack Splinter, only for the rat to drop him on his ass.
Bebop and Rocksteady hit the Museum of Natural History and steal the next piece for the portal machine.
Donnie analyzes a sample of the mutagen and discovers it has the ability to possibly turn him and his brothers human. He tells Leo, but he thinks it would be a bad idea, and he tells Donnie not to let the others know. Mikey heard the whole thing and he tells Raph. Raph confronts Leo for keeping secrets from his brothers. Raph and Mikey think it would be great if they could be human so that they could walk among the humans without being seen as freaks, but Leo insists that they are meant to be turtles.
With April and Casey's help, Mikey and Raph try to sneak into the police station to recover the mutagen sample while Leo and Donnie are on another mission. When Mikey and Raph encounter trouble, they call their brothers for help. The police corner the Turtles, with one of the cops calling them monsters. April and Casey run in to cover them, leaving them to get arrested while the Turtles retreat. Leo chastises his brothers, saying they do not work well as a team. He goes to voice his concerns to Splinter, who says that the fact that each Turtle thinks differently is what makes them a strong team.
The Turtles learn that Bebop and Rocksteady are headed to Brazil to recover the last piece of the machine. The thugs find the piece and are already flying back toward New York. The Turtles, flying in their own plane, drop down to intercept them. They jump off their plane and enter the villains' plane. They encounter Bebop and Rocksteady face-to-face and begin fighting them. Rocksteady hops inside a tank and shoots at the Turtles, unintentionally causing the plane to crash into the river. Rocksteady continues firing from the tank as the Turtles still try fighting him and Bebop. The Turtles then plummet down a waterfall, but they hide in their shells for safety.
Back in New York, April and Casey are being detained by Vincent. Vincent plays security footage from Stockman's lab to make it look like April broke in and stole the mutagen sample. Casey manages to pick Vincent's pocket to get her phone to let April call Vern. Vern uses his status to get into the TCRI labs and steal the unaltered security footage. He brings it to the police station and shows it to Vincent. The video then shows the mutated Bebop and Rocksteady to prove April and Casey aren't crazy. Vincent has no choice but to let them go.
The villains now have each piece to the portal machine, which Stockman uses to open the portal to Dimension X. Shredder betrays Stockman and has his ninjas take him far away. Shredder returns to Dimension X and tells Krang that everything is falling into place. However, Shredder gets a taste of his own medicine when Krang says he plans on taking all the glory in his scheme. Krang freezes Shredder and holds him within his ship before making his way to Earth.
The Technodrome begins forming piece-by-piece above the city. The Turtles decide they must spring into action, but are conflicted on whether or not they do it as humans or turtles. They take the mutagen sample they got, but Raph chucks it against the wall so they can take Krang on as NINJA TURTLES. On the surface, they are met by April, Casey, Vern, Vincent, and the cops. Vern then admits that he got help from his friends in taking down Shredder. Vincent meets the Turtles and becomes certain that they mean to save the city.
The Turtles hop on the flying pieces and find Krang on his ship. As they fight him, April and Casey confront Karai at the lab. The Turtles bring Krang and his robot down while April takes Karai out, and Casey closes the portal. The Technodrome falls apart, bringing it and Krang back to Dimension X. Krang vows to return for the Turtles, bigger and stronger than before.
The Turtles are honored by Vincent and the NYPD for their heroics. April, Casey, Vern, and even Splinter are their to support them. Although they are given the chance to live above the sewers, the Turtles decide that they will stay where they are, and they will continue to protect the city. In the final shot, the brothers then go to the top of the Statue of Liberty and cheer for their victory.