A group of vampires look for the hidden resting place of "Drake", also known as Dracula, the genetic "father" of their species. They find and wake him in a Syrian ziggurat tomb, although not before, in a moment of blood lust, he kills one of them. The vampires capitalize on Blade's reputation as a serial killer and frame him for the killing of a familiar. During the ensuing manhunt, the FBI locate Blade's latest hideout, and, during the siege, Abraham Whistler dies. With his mentor gone and surrounded by human authorities, Blade allows himself to be captured and arrested.
After both medical and interrogative sessions prove futile, the police prepare to hand Blade over to a group of medics, who are, unknown to them, vampires. He is rescued by Hannibal King and Abigail Whistler, Abraham's daughter, both of whom belong to a group of vampire hunters called the Nightstalkers. King and Abigail reveal that the vampire Danica Talos, who temporarily turned King into a vampire, has located Drake. Because he is the first of their kind, Drake is invulnerable to sunlight. Talos hopes Drake is powerful enough to kill Blade and make the rest of the vampires into daywalkers as well.
The Nightstalkers believe the vampires are too numerous to kill individually, so Sommerfield, their resident chemical specialist, has created an experimental bioweapon dubbed Daystar, an airborne virus capable of killing every single vampire in the world on a molecular level.
In their first confrontation, Drake tests Blade by drawing him away from King and Abigail, incapacitating King in the chaos. Although physically superior, Drake shows an affinity with Blade, as they are both unique to their race and "honorable warriors" by choice. While Drake converses with Blade, he uses a baby that he has taken hostage as leverage. Drake considers all humans unworthy of his notice and holds his own followers in contempt. Blade and Abigail learn of the vampires' planned "final solution": blood farms where brain-dead humans are harvested for their blood. Blade shuts down the victims' life support in an act of euthanasia.
The two return to find all the Nightstalkers but King and Sommerfield's young daughter, Zoe, dead; Drake uses the two survivors to lure Blade into combat. A posthumous recording by Sommerfield reveals that she has perfected a small quantity of the Daystar virus, but it requires the blood of Drake and may kill Blade. Meanwhile, King is chained and tortured by Jarko Grimwood and Asher Talos for information about Daystar. When this fails, Danica Talos, tortures King by pressuring his covered wound with the tip of the high heel of her shoe. She also threatens to turn King into a vampire again and force him to feed on Zoe.
Blade and Abigail arrive and fight the Talos' underlings and the building security force. After freeing both King and Zoe, Abigail kills Asher, King kills Grimwood, and Blade engages Drake in a sword battle. In the end, Blade impales Drake with Abigail's Daystar arrow, which releases the virus into the air, killing all the nearby vampires, including Talos. As Drake dies, he praises Blade for fighting with honor and tells him that through Blade, the vampire race will survive. Dying, he offers Blade a "parting gift"; he also warns him that, "sooner or later, the Thirst always wins".
From here there are multiple endings:
Theatrical ending: As Blade fought honorably, Drake gives him a "parting gift" by transforming his dying body into a replica of Blade's. The FBI retrieves the body of who they think is Blade, and thus call off their manhunt. Just as they begin the autopsy, Blade's body reverts into that of Drake's. Hannibal's voiceover tells the viewer that Blade is still alive somewhere, finishing the last struggles of the war; having rejected Drake's hopes of prolonging the vampire race, Blade's war will never end.
Unrated ending: The body retrieved by the FBI is Blade's, but he's not really dead. Drake's body is nowhere to be seen, hinting at his survival. At the morgue, Blade sits up abruptly, attacks the FBI agents, and appears ready to bite a nurse on the neck. The ending is ambiguous as to whether Blade has retained his humanity or given in to his vampire thirst as Drake predicted. This is the ending seen on the director's cut of the film, and commentary on the DVD indicates it was the ending director Goyer intended.
Werewolf ending: The Daystar virus circles the globe and wipes out all vampires. Blade walks off into the sunset, his long battle finally over. The final shot is of the Nightstalkers battling a new enemy: werewolves. This version of the ending was used in the novelization of the film and is included on the DVD as an extra.