Over the two years since the death of Deacon Frost, Blade (Wesley Snipes), the Daywalker, a vampire unaffected by sunlight and who chooses to defend humans and fight other vampires, has been trying to find his mentor Whistler (Kris Kristofferson), who survived his suicide attempt in the first crisis. Blade has been sweeping across Russia and eastern Europe, enlisting the aid of a young man, Scud (Norman Reedus), to design him a new line of gadgetry and weaponry. Blade carves his way through a large gang of vampires, sparing one, Rush (Santiago Segura), when he promises to reveal Whistler's location. Blade tells Rush he will be back for him. Blade finds Whistler locked in a tank by a gang of vampires who were keeping him alive to torture. Blade rescues Whistler and brings him back to the lair.
Meanwhile, a crisis has arisen in the vampire community. A mutated strain of vampirism (dubbed the "Reaper virus") is sweeping through their ranks, giving its carriers new characteristics. The original carrier of the strain is Jared Nomak (Luke Goss), who attacked the blood bank at the beginning of the film. Stronger than common vampires, the Reapers have three-way jaws, leech-like sucker tentacles, and hearts encased in a thick layer of bone, making them almost invulnerable to any weapon barring sunlight. They also have a ravenous hunger, requiring more feeding than vampires, and anyone they bite, Human or Vampire, become more Reapers, even if they died while being fed upon. In order to combat the mutants, the vampire overlord Eli Damaskinos (Thomas Kretschmann) sends his minion, Asad (Danny John-Jules), and daughter Nyssa (Leonor Varela) to find and strike an uneasy truce with Blade. Upon meeting Damaskinos and his familiar Karel Kounen (Karel Roden), Blade learns of the vampire community's plight. They concede to him that the Reapers are the greater evil and once they finish the vampire population, they will descend on humankind.
To this end, Blade teams up with the Bloodpack, a group of vampire warriors and assassins who were originally assembled to kill him. The group consists of Reinhardt (Ron Perlman), Me Chupa (Matt Schulze), Snowman (Donnie Yen), Verlaine (Marit Velle Kile), her lover Lighthammer (Daz Crawford), and Priest (Tony Curran). Blade plants an explosive on the back of Reinhardt's head to keep him under control.
The group starts at a vampire nightclub, while Whistler acts as a sniper to guard Scud in the team's transport. The mission goes wrong when the group finds the Reapers are immune to silver, are physically stronger than normal vampires, and resistant to normally crippling injuries even for vampires. Priest is fed on by one of the Reapers, subsequently transforming into one and being mercy-killed by the rest of the Bloodpack, and Lighthammer is bitten, but conceals it. Meanwhile, Blade fights Nomak, who proves immune even to Blade's anti-coagulant spikes, and in the end, after a stalemated battle, Nomak leaves only because of the sunlight.
While Blade and the Bloodpack are fighting in the club, Scud is attacked by several Reapers, while Whistler disappears. Scud is able to drive off the attacking Reapers using UV lights on the transport. Whistler returns after the battle to show them a dying Reaper at a sewer entrance, revealing the location of the Reaper Nest. Nyssa autopsies the dying Reaper while Scud and Whistler make ultraviolet weapons for the team.
Upon entering the Reaper nest, Lighthammer succumbs to the bite he received at the club and transforms into a Reaper, subsequently killing Snowman. He then chases down Verlaine who kills them both when she opens a sewer hatch to the sunlight. Chupa and Reinhardt decide to punish Whistler for Priest's death, but Chupa is killed when Whistler sprays him with a Reaper pheromone, which attracts the horde. Asad and Nyssa walk into a nest and Asad is pulled underwater and killed. Nyssa is saved by Blade, who uses a cluster-UV device to destroy all the Reapers except Nomak, while Nyssa and Reinhardt escape the blast.
After the battle Blade is betrayed and captured by Damaskinos' forces, along with Whistler and Scud. It is revealed that, in his efforts to create a new race of vampires immune to sunlight and silver, Damaskinos turned his own son, Nomak into the first Reaper. Scud is also revealed to be one of Damaskinos' familiars. Blade however, has been aware of Scud's status since the beginning of their partnership, and kills him with Reinhardt's bomb after Scud reclaims it, believing it was a dud. With the Reapers gone, Damaskinos plans to dissect Blade to learn how to create more Daywalkers. However, Whistler escapes and rescues Blade. Blade fights his way through Damaskinos' henchmen, which ends with Reinhardt being killed.
Meanwhile, Nomak has entered Damaskinos' stronghold seeking revenge on his father. Nyssa, having become disillusioned with her father's extreme methods, betrays Damaskinos by sealing off their escape route to the heliport. Damaskinos is killed by Nomak after trying to negotiate with him. In order to "complete the circle", Nomak bites Nyssa, drinking her blood and infecting her with the Reaper virus. When Nomak attempts to leave, he is then confronted by Blade. After a brutal fight, Blade finds the weak spot in Nomak's physical defenses and jams his sword beneath Nomak's arm, bypassing the bone shield around his heart and mortally wounding him. With his revenge complete, and wanting the suffering of his mutation to end, Nomak then willingly pushes the sword the rest of the way through, killing himself. With Nomak dead, Blade carries Nyssa outside for the sunrise before she transforms, in order to honor her last wish: to see the sunlight for the first time, and die as a vampire.
With the crisis over, Blade tracks down Rush to a strip club in London and finishes him off.