1887, Transylvania, Doctor Frankenstein (Samuel West) brings to life his Monster (Shuler Hensley) with the aid of his assistant Igor (Kevin J. O'Connor), and Count Dracula (Richard Roxburgh). Dracula kills Frankenstein after revealing that he helped him only so he could use the Monster to bring his undead children to life. The Monster then escapes to a windmill, which is burned down by a pursuing mob. The mob flees as Dracula and his three brides, Verona (Silvia Colloca), Aleera (Elena Anaya) and Marishka (Josie Maran), mourn the loss of the Monster and their chance to bring their children to life.
One year later, the Knights of the Holy Order, stationed at the Vatican, dispatch Gabriel Van Helsing, who has amnesia, to kill Dracula. He is also tasked with preventing the last of the Valerious family from falling into purgatory; the family swore to kill Dracula nine generations ago and is unable to enter Heaven until they succeed. He is given a torn piece of paper with an insignia on it. He is joined by Carl (David Wenham), a friar who provides support and weapons.
Arriving in Transylvania, the two meet Anna Valerious (Kate Beckinsale), who tells them her brother Velkan (Will Kemp) was recently killed by a werewolf. Van Helsing then saves her from Dracula's brides as they attack the village, ending with Van Helsing killing Marishka as the others escape. Anna then takes the pair back to her castle. Anna is determined to kill Dracula herself, but Van Helsing is unwilling for her to take the risk, knowing that she is the last of the Valerious family. When she resists, he gases her to sleep and puts her in her bed.
Later in the night, Anna awakens from her deep, dreamless sleep and encounters Velkan, now a werewolf himself. After Velkan flees, Van Helsing and Anna track him to Frankenstein's castle, only to find Dracula attempting to give life to his children using Velkan as a substitute for the Monster. Anna frees Velkan but he becomes a werewolf again. Dracula confronts Van Helsing, who recognizes him from his past.
While escaping, Van Helsing and Anna fall into a cave. There, they find Frankenstein's Monster alive. Van Helsing decides to take him to Rome so he can be protected. They flee in a carriage, but while crossing the Carpathian Mountains, the brides and Velkan, who have been pursuing the group, attack. The carriage plummets down a precipice and Verona tries to save the Monster, but on opening the door reveals that it is a decoy carriage containing only stakes bundled against explosives, which kill her when the carriage hits the bottom.
The genuine carriage is attacked and Velkan is killed by Van Helsing, but not before Van Helsing is bitten by him; when the next full moon occurs, Van Helsing will become a werewolf. Anna is then captured by Aleera and taken to Budapest. In Budapest, Van Helsing agrees a trade and hides the Monster before he and Carl head off to save Anna, who is at a masked ball for vampires. They manage to rescue her, but the Monster is captured and taken away on a boat. Escaping from Dracula's Summer Palace, Van Helsing, Anna, and Carl return to Frankenstein's castle, where they find all the equipment has been removed. At Anna's castle, Carl explains that Dracula was the son of Anna's ancestor. Dracula was murdered, but not before making a Faustian Bargain, which gave him new life as a vampire. Carl explains that although Anna's ancestor made the vow to kill Dracula, he couldn't kill his own son. Instead, he banished Dracula to an icy fortress from which he should not have been able to return, but the Devil gave him wings and the power of flight, which allowed him to escape. Van Helsing then finds a portal to Dracula's castle disguised as a wall map, completed using the paper that Van Helsing brought from Rome. They enter the portal, emerging on a cliff near Castle Dracula.
As the trio sees the Monster being lifted to the laboratory, he tells them that Dracula has a werewolf cure. Carl realizes that only a werewolf can kill Dracula and that he uses werewolves to do his bidding, but needs a cure in case they have the willpower to turn against him. Making his way to the laboratory, Van Helsing frees the Monster, then becomes a werewolf and enters a final battle with Dracula (who turns into a giant bat-like creature). Anna and Carl retrieve the cure but are attacked by Aleera and Igor. Igor falls to his death off a bridge, and Aleera gets impaled by Anna with a silver stake. Dracula reveals that Van Helsing is really Gabriel, the Left Hand of God - as well as the one who originally murdered him. He offers to restore Van Helsing's memories, but Van Helsing refuses, deciding that "some things are better left forgotten". He then bites into Dracula's throat, killing him and his offspring. Anna arrives with the cure and injects Van Helsing with it, only to be killed by him at the same time, much to his grief.
Van Helsing and Carl hold a quiet ceremony for Anna and cremate her as the Monster departs on a raft into the ocean, having been allowed a chance at life. As Anna's body burns, Van Helsing sees her and her family in Heaven, at peace thanks to Dracula's death. This sight cheers Van Helsing up as he and Carl depart for their next adventure.