While Leonardo, Raphael and Donatello all have brown eyes Michelangelo has blue eyes. According to the film's director this was done to make Michelangelo seem a bit more innocent and younger than the other turtles.
Towards the end of the film, several items are shown that reference the live action movies. These include: The Shredder's helmet and staff, a Foot Ninja Mask, the canister of ooze that mutated the turtles and Splinter (labeled TCRI instead of TGRI, though a crack is strategically placed to work either way), the Time Scepter, Walker's hat, Lord Norinaga's helmet, and pieces of the suits of armor worn by the Turtles in the third film.
Specific dialog makes references to the previous live-action films. In the battle with the Foot Clan, Casey Jones says, "Two minutes for high sticking!" which was one of his lines from his introduction in the same film. The very last line of the film, spoken by Raphael is, "Man, I love being a turtle!" which has been spoken in each of the previous films.
Splinter's appearance is generally consistent with the previous movies (in which he is depicted as having brown fur), although in this movie, he wears a proper karate gi, as opposed to dirty rags from the previous movies. His right ear is also no longer cut as it was in previous movies. Kevin Munroe jokingly responded that Splinter had an otoplasty.
Some of the 13 monsters are based on actual 'monsters' from popular myth - the first one that appears in the unfinished building is based on the Yeti, the next one to be captured is obviously the Cyclops, the one that eats a pigeon on a building is clearly modeled on gargoyles, and the one in the diner brings to mind the Jersey Devil.
Jim Cummings is the only Teenage Mutant Ninja Turtles actor to make it into this movie since his first TMNT appearance in the 1987 version of the classic TMNT cartoon series, which he played Leatherhead, Genghis Frog, and the 1991 alternate of Shredder.
As revealed on the director's commentary on the DVD one of the early drafts of the script had Raphael briefly dying and Donatello using Winter's technology to bring him back to life. This idea was turned down by Peter Laird however who disliked the idea of having one of the turtles dying.
During the fight between the Nightwatcher and the monster in the diner, at one point, a refrigerator falls on top of the monster and the music cuts out and the monster is heard making a whimpering noise. This was actually a mistake in the soundtrack, but when the filmmakers saw it, they couldn't stop laughing and left it in the film.
During the first Nightwatcher scene, when the criminals are fleeing, one of the signs on the shops in the background spells out "monro" in hiragana, a Japanese writing system, which is most likely related to the writer and director Munroe.