Denisof was born in Salisbury, Maryland, to a Russian-American father and an Irish-American mother, Christiana. He also indicates that he is a small part French on his father's side. He moved to Seattle, Washington when he was three years old. After graduating from St. Paul's School in Concord, New Hampshire, he moved to Britain where he lived and worked for a number of years.
He was the fight director for a stage production of Hamlet, and in 1991 he was the fight director of The Other Side for the BBC, Romeo and Juliet, and The Soul's Dark Night. One of his first known works in television is on the arcade version of the video for "Got My Mind Set On You" by George Harrison. He had a small role in the movie First Knight appearing in scenes with Sean Connery and Richard Gere.
Denisof appeared with Sean Bean in three episodes of Sharpe as Lord John Rossendale. He has done many stage productions, including one with Anthony Stewart Head, who played Wesley's predecessor Rupert Giles on Buffy the Vampire Slayer.
Denisof's character was first introduced in Buffy the Vampire Slayer season three as Buffy's replacement Watcher. He later showed up in episode 10 of season one of Angel, and was an official main character from episode 11 (effectively replacing Doyle), appearing in 100 out of the 110 episodes of the series.
Denisof was among 200 actors considered for the role of James Bond for Casino Royale, but lost out to Daniel Craig. He also auditioned for lead in Highlander: The Series back in the early 1990s.
Denisof appears in Season 2 of Private Practice as a man named Daniel, who has two pregnant wives giving birth on the same day.
In 2006, Denisof began a recurring role as Sandy Rivers on Alyson Hannigan's series How I Met Your Mother and has appeared in 7 episodes to date, most recently being seen on the April 16, 2012, episode. According to a commentary in season 1, creators Craig Thomas and Carter Bays speculated that Denisof was initially reluctant to take the role because he feared it was just charity work on account of his wife, Hannigan, being a main character on the show. They go on to state that he was not aware, until they actually told him, that they were huge fans of his work, dating back to his time on Angel.
In 2012, Denisof reunited with Joss Whedon again in The Avengers as The Other, the servant to the character Thanos.
According to the DVD release of Angel's Season Five, three weeks before the filming of the season premiere Denisof was stricken with Bell's palsy, from which he later recovered. Due to the paralysis of the left side of his face, scenes were structured as to not show its effects.
Denisof with wife Alyson Hannigan. Denisof married past co-star Alyson Hannigan, who played Willow Rosenberg in Buffy the Vampire Slayer, on October 11, 2003. The couple lives in Santa Monica. Their first child, a daughter named Satyana Marie Denisof, was born on March 24, 2009 (which is also Alyson Hannigan's birthday). Their second daughter, Keeva Jane Denisof, was born May 23, 2012.
Denisof has appeared in both Buffy the Vampire Slayer and its spin-off Angel.