In 1967, a pregnant woman is attacked by a vampire, causing her to go into premature labor. Doctors are able to save her baby, but the woman dies of an unknown infection.
Thirty years later, in 1997, the child has become the vampire hunter, Blade. He raids a Los Angeles rave club owned by the vampire Deacon Frost. Police take one of the vampires to the hospital, where he kills Dr. Curtis Webb and feeds on hematologist Karen Jenson and escapes. Blade takes Karen to a safe house where she is treated by his old friend Abraham Whistler (Kristofferson). Whistler explains that he and Blade have been waging a secret war against vampires using weapons based on their elemental weaknesses, such as sunlight, silver, and garlic. As Karen is now “marked” by the bite of a vampire, both he and Blade tell her to leave the city.
Meanwhile, at a meeting of the House of Erebus, a council of vampire elders, Frost, the leader of a faction of younger vampires, is rebuked for trying to incite war between vampires and humans. As Frost and his kind are not pure-bloods (natural-born vampires), they are considered socially inferior. In response, Frost has one of the elders executed and strips the others of their authority.
Upon returning to her apartment, Karen is attacked by police officer Krieger, who is a “familiar”, a human slave controlled by a vampire. Blade subdues Krieger and uses information from him to locate an archive that contains pages of the Book of Erebus (the vampire bible); he comes upon Pearl, a morbidly obese vampire and tortures him with a UV light into revealing that Frost is planning to conduct a ritual to summon the vampire blood god, La Magra. Later, at the hideout, Blade injects himself with a special serum that suppresses his urge to drink human blood. However, the serum is beginning to lose its effectiveness due to overuse. Krieger informs Frost of what happened, and Frost kills and eats Krieger.
While experimenting with the anticoagulant EDTA as a possible replacement, Karen discovers that it explodes when combined with vampire blood. She manages to synthesize a vaccine that can cure the infected but learns that it will not work on a human-vampire hybrid like Blade. Karen is confident that she can cure Blade’s bloodthirst but it would take her years of treating it. Frost and his men attack the hideout, infect Whistler, and abduct Karen. When Blade returns, he helps Whistler commit suicide and arms himself with special syringes filled with EDTA.
When Blade attempts to rescue Karen from Frost’s penthouse, he is shocked to find his still-alive mother, who reveals that she came back the night she was attacked and was brought in by Frost, who appears and reveals himself as the vampire who bit her. Blade is then subdued and taken to the Temple of Eternal Night, where Frost plans to perform the summoning ritual for La Magra. Karen is thrown into a pit to be devoured by Webb, who has transformed into a decomposing zombie-like creature. Karen injures Webb with a sharp bone and escapes. Blade is drained of his blood, but Karen allows him to drink from her, causing him to recover. Frost completes the ritual and obtains the powers of La Magra. Blade confronts Frost after killing all of his minions, including Blade’s mother. During their fight, Blade injects Frost with all of the syringes, causing his body to inflate and explode, killing him.
Karen offers to help Blade cure himself, but he asks her to create a new serum he can sustain on instead, as he intends to continue his crusade against vampires. In a brief epilogue, Blade confronts a vampire in Moscow.