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Luke Valentine was one of the artificial vampires Millennium Organization used against the Hellsing Organization, along with his foul-mouthed brother Jan. While he is a part of the top tier of Millennium's finest officers, the Werwolf team, his brother received no such distinction. He held the rank of Warrant Officer, putting him in the same level as Schrodinger. He held the "honor" of being the leader of Millennium's first strike team led against Hellsing, and the first to officially being entrusted with the mission to kill Alucard.

Luke, dressed in higher-end smart white clothes, developed the plan to destroy Hellsing and the Convention of Twelve by assaulting the building with an army of ghouls, which fed upon the fallen Hellsing commandos, simultaneously turning them against their former masters and giving the invaders extra bodies to throw against the defenders. The commander of these ghouls would be Jan, while Luke ventured further into the basement to single-handedly fight Alucard. His unnerving speed, superior to even that of fellow Millennium officer Tubalcain Alhambra, allowed him to quickly dispatch the Hellsing soldiers with a recurved bowie-knife and fight his way to the basement.

There, he met Alucard. Both were initially corteous, but then both started fighting seriously, Alucard with the Jackal, Luke with a set of sawn-off rifles. Alucard appeared to spectacularly fail to hit Luke, while in fact he was using the Jackal's much larger bullets to cause the exit tunnel to collapse. Then, Alucard declared to be impressed by Luke and released his control art restrictions to Level One. This allowed him to shift into a far more powerful form, capable of warping the ambience of the fight. Changing the scenery into a dark, hellish world, he used the Jackal's bullets to shoot off Luke's legs. When he stood poised to stike the deathblow against Luke after consuming one of his legs, Alucard taunted him, and challenged him to pull a power feat similar to his own. Luke called him a monster, causing Alucard to realize exactly how weak Luke really was. He then had his hellhounds devour Luke and said of him, representing his opinion on the whole race of artificial vampires: "As a vampire, you were nothing but shit. Now you are nothing but dog shit."

He made a surprise reappearance during the final stages of the London assault, when former Hellsing trashman turned Millennium assassin, Walter C. Donez, managed to rip open Baskerville, Alucard's hellhound. Luke emerged from the remains, frankly disgusted at what he saw. Later, Walter attempted to deliver the deathblow against Alucard by holding him with his wires and impaling him upon a piece of rebar. However, the "Alucard" Walter killed thus was nothing but a decoy created from Luke's remains, costing him the time he needed to slay Alucard and losing him the battle.

In the anime, he appears as a more cultured thug, but is slain in the same brutal manner as in the manga and OVA. He owned a somewhat seedy nightclub and had some underworld ties in this version. He also attempted to weaken Hellsing before he struck with his army of ghouls, having one of Jan's prostitutes serve as bait for Hellsing operatives in a rigged trap that would have killed many of them.

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