María LaGuerta (Lauren Vélez) is promoted to Captain after blackmailing Deputy Chief Matthews (Geoff Pierson), whose name was on a prostitute's ledger. Vince Masuka (C.S. Lee) is teaching a group of forensic science students, and eventually asks a female student, Ryan Chambers (Brea Grant), to become his intern, only to have to fire her when she steals old evidence from the department: a painted prosthetic hand from the Ice Truck Killer crime scenes, which shows up on an Internet auction site. Masuka quickly hires another intern, video game designer and computer programmer Louis Greene, and begs him to fix the problem. Greene claims he got the site to remove the auction but was unable to get the hand back.

LaGuerta and Angel Batista (David Zayas) have divorced, but try to remain friends; this is further complicated by Matthews' decision to promote Debra Morgan (Jennifer Carpenter) to LaGuerta's vacant lieutenant position instead of Batista, who thinks this is a result of the feud between Matthews and LaGuerta. In retaliation, LaGuerta continuously thwarts Debra's attempts to make a good first impression as the new lieutenant by giving her the wrong advice; however, Debra makes an independent decision to hire Mike Anderson (Billy Brown) as her replacement, against LaGuerta's recommendation.

Batista's sister Jamie (Aimee Garcia) has become Harrison's babysitter, and Dexter and Debra visit a Catholic pre-school in hopes of signing Harrison up. Quinn (Desmond Harrington) proposes to Debra, but she refuses and they break up. Shortly after, Quinn learns of Deb's promotion and surmises that was her reason for ending their relationship. Quinn begins a long pattern of barhopping, drunkenness, and one-night stands; his unprofessional behavior angers and ultimately endangers Batista, his new partner.

This season features two main antagonists who become known as the Doomsday Killers (or DDK), Professor James Gellar (Edward James Olmos) and his student Travis Marshall (Colin Hanks), who seek to bring about the end of the world through killings based on the Book of Revelation. They leave signs of the Apocalypse including the Alpha and Omega or the Four Horsemen as a cryptographic tableau at crime scenes. The season also introduces Brother Sam (Mos Def), a former drug addict and murderer who repented and became a minister. He operates a body shop where he employs other ex-convicts, whom he also "shepherds" to lead crime-free lives.

Initially believing Sam to be behind the first Doomsday Killer murder, Dexter is quickly proven wrong and finds himself befriending him. Dexter finds new light and guidance from Brother Sam in being a better father to Harrison and in keeping his Dark Passenger at bay. However, Brother Sam is murdered by one of his trusted ex-convicts; Dexter learns who did this and, going against Brother Sam's wish to forgive his killer, kills the murderer himself.

Dexter learns that the Trinity Killer's wife and daughter have been found dead in Nebraska, which Jonah Mitchell reports was the work of his father, Arthur Mitchell. Dexter, the only person who knows the Trinity Killer is dead, suspects that Jonah is following in his father's footsteps and goes to Nebraska to kill Jonah, encouraged by a subconscious vision of his brother, Brian Moser, the Ice Truck Killer. However, after confronting Jonah, Dexter learns that his sister committed suicide and Jonah killed his mother unintentionally, and now wants to die. Dexter decides to forgive Jonah and leaves him alive to deal with his demons.

Dexter's investigation of the Doomsday Killers leads him to Travis Marshall. Travis says that all he has done was at the request of Professor Gellar, so Dexter asks Travis to help him kill the Professor, thinking he can save Travis as Brother Sam tried to save him. However, Dexter eventually discovers that Professor Gellar has been dead for years and now exists only in Travis' mind. Travis marks Dexter as "the Beast" and tries to kill him in one of his tableaus, the Lake of Fire, but Dexter escapes and is saved by a passing migrant boat. Finally, Travis kidnaps Dexter's son to use as a sacrifice in his final tableau, thinking that "the Beast" is dead. Dexter rescues Harrison and knocks Travis unconscious. He takes Travis back to the church where Travis carried out his earlier murders, which Dexter has set up as a kill room. As Dexter prepares to kill his victim, Debra enters the church and sees Dexter plunge a knife into Travis' chest.

The season's subplots include Angel's sister dating Louis Greene, who wants to impress the police force, especially Dexter, and is revealed to have acquired the prosthetic hand from the Ice Truck Killer case, which he mails to Dexter after marking it up with a certain pattern. Debra refuses to yield to pressure to close the case of the overdose death of a prostitute, eventually discovering that Chief Matthews was present when the woman died. Matthews is fired after LaGuerta spreads the information. Finally, Debra attends department-ordered therapy sessions after being involved in a shootout, and during her sessions, begins to think/has the idea planted that she may have romantic feelings for Dexter.