Four co-eds are about to graduate from a secret academy, where they have been trained since high school to become spies in the paramilitary group D.E.B.S. (Discipline, Energy, Beauty, and Strength). Candidates are selected through a test hidden in the SATs that measures an applicant's ability to fight, cheat, lie, and kill. The team is headed by the somewhat obsessive Max (Meagan Good), who tends to draw her gun at the slightest provocation. The other team members are the prissy and somewhat naive Janet (Jill Ritchie), the chain-smoking and promiscuous French-exchange student Dominique (Devon Aoki), and Amy Bradshaw (Sara Foster), who is doubtful about being a spy, despite the fact that she earned a perfect score on her D.E.B.S. test and is now an honor student at the academy. Amy is considering going to art school in Barcelona after graduation, but Max, who has been her best friend since freshman year, tries to convince her that espionage is more important.
As the film opens, Amy has broken up with her boyfriend Bobby, a Homeland Security agent. Her personal life takes a back seat to the re-emergence of Lucy Diamond, a criminal mastermind whom, by coincidence, is the subject of Amy's thesis. The D.E.B.S. are sent by the head of D.E.B.S., Miss Petrie (Holland Taylor), to surveil Lucy Diamond (Jordana Brewster). They arrive at the restaurant Les Deux Amours expecting to witness criminal dealings. Little do they know that Lucy Diamond has simply been set up on a blind date by her chief henchman and best friend, Scud (Jimmi Simpson). She is there to meet an ex-KGB assassin named Ninotchka Kaprova. The date is a disaster. Bobby shows up to confront Amy about their breakup, but accidentally alerts Lucy Diamond to the spies hanging on swings overhead. A shootout erupts, and Diamond escapes in the chaos. The D.E.B.S. follow her, splitting up into two groups with Max and Dominique going one way, and Amy and Janet going the other. Amy and Janet get separated and Amy literally runs into Lucy in a large storage room. They quickly end up in a standoff, with Amy trying to convince Lucy to come quietly. Lucy instead charms Amy (who is surprised to learn Lucy is a lesbian, since none of the material she studied for her thesis revealed this) into lowering her guard and disappears the moment Amy's attention wavers. When the other D.E.B.S. arrive, they praise Amy for being the only federal agent who has ever encountered Lucy and lived to tell about it.
Despite Scud's protests ("Not only is she a D.E.B., She's the D.E.B.! She's the perfect score!"), Lucy returns to the squad's residence that night to talk to Amy, with whom she is quite taken. They fight, until Lucy gains the upper hand and forces Amy to go out with her for a night of fun. However, they encounter Janet outside the house. Lucy has no choice but to bring Janet along or risk discovery. They drive to an underground nightclub called 'The Junk Pit', which is reached by driving through holographic barriers disguising it from casual observation. Amy has every intention of sulking in the car, but Janet goes inside to use the bathroom and Amy accompanies her.
Inside the club, Lucy notices Amy and convinces her to have a drink. Janet ends up bonding with Scud while Lucy further charms Amy. Lucy is intrigued by Amy's intellect, her breakup with Bobby, and her obvious doubts about being a spy. Amy is fascinated to discover that much of Lucy's deadly reputation has been carefully manufactured and that many if not all of the agents who supposedly died while confronting her actually died from environmental factors like frostbite and Ebola. Lucy senses Amy's attraction and leans in to kiss her only to be interrupted by a shocked Janet and a bemeused Scud.
Lucy takes the two D.E.B.S. home. Janet is indignant, claiming that Amy is violating protocol, and is further troubled at learning Amy might be bisexual. Amy blackmails her into silence by reminding her of an incident where Janet "got drunk on peach wine coolers, and nearly got the chancellor of Bulgaria killed" and Amy covered for her. The next day, Miss Petrie meets with Amy and promotes her to squad leader, effectively replacing Max. Max is furious, but must follow Amy as they respond to a bank robbery orchestrated by Lucy.
At the bank, Amy and Max bicker over the best approach to rescuing the hostages and capturing Lucy Diamond, who, despite Scud's disapproval, has committed the crime solely for an opportunity to see Amy again. Amy rushes blindly forward and the squad gets trapped inside the bank vault. A chute opens under Amy's feet; she falls out of the vault, leaving the other three behind. Amy arrives in the bank's basement, where Lucy is waiting for her. Meanwhile, the vault has turned into a death trap as the ceiling sprouts large spikes and begins to descend. In the basement, Amy tells Lucy that they cannot be together and gets her to release her squadmates, although, spiked ceiling notwithstanding, Lucy claims her squadmates were never in danger of death, and releases them from the vault. Lucy attempts to cover her interest in order to salvage her pride and turns to leave, but is pulled back by Amy, who can no longer resist her attraction to Lucy. They kiss, and Lucy asks Amy to come with her. The D.E.B.S. arrive in the basement to find Lucy's callsign, diamonds, Amy's tie, and a note spraypainted on the wall reading "I HAVE THE GIRL." They assume Amy has been kidnapped, though Janet seems to know exactly what has happened.
Miss Petrie organizes a nationwide manhunt to find Amy while she spends some quality time with Lucy; Janet desperately tried to keep Amy apprised of the situation, but Amy never hears any of Janet's phone messages. Amy shares her art school dream with Lucy, who suggests running off to Barcelona together, where Amy can attend art school and Lucy will rent sailboats to tourists. Lucy and Amy are seen by a jealous Ninotchka, who anonymously tips Max to their location.
Lucy and Amy are arguing about their respective professions while the three D.E.B.S. and Bobby close in on Lucy's lair. After a gun battle between Scud and his henchment, they continue onward, and catch the two women in a moment of passion. Mortified, Amy returns to the D.E.B.S and attempts to explain herself. Miss Petrie is ready to exile her, but Max steps in with an alternative to save Amy's reputation and thus prevent the D.E.B.S from looking foolish for having held her in such high academic esteem. Amy will tell everyone that she was brainwashed by Lucy, but that the D.E.B.S. rescued her in the nick of time. Tearfully, Amy agrees to comply. Max is still disgusted with Amy, and Amy finds herself being shunned by her friends for her deception. When the academy headmaster, Mr. Phipps, tries to encourage her on her upcoming graduation, she is further distressed to learn her perfect D.E.B.S. test score meant that she was "a perfect liar."
Meanwhile, Lucy does everything she can to win Amy back, returning all the money she has ever stolen and projecting a valentine message into the sky like a Bat-Signal. Finally, she and Scud plan to crash the D.E.B.S. year-end dance, Endgame, where Amy is to be made D.E.B. of the year. At the dance, Lucy gets spotted by security guards and must evade Bobby while Amy gives her speech, unaware of Lucy's presence. Lucy fights her way to the auditorium, where she is just in time to hear Amy publicly condemn her. She is saddened, but catches Amy's eye and smiles hopefully. Amy retracts her entire speech, and admits that the days she spent with Lucy were the best days of her life, before running offstage. She and Lucy find each other in a storage room very like the one in which they first met, and they share another long-awaited kiss. Max, Janet, and Dominique catch up to them, guns raised. But Max has by this time reconciled herself with the fact that her best friend is in love. She lets them go and misdirects Bobby and the security squad so that Amy and Lucy can make their escape. Scud then starts to ask something of Janet, who agrees before he has even finished speaking.
The final scene has Lucy and Amy driving off together into the beautiful night.