The recognition, for the British-born actor's performance in the title role of Steven Spielberg's biopic of US President Abraham Lincoln, puts him above Hollywood legends including Dustin Hoffman and Marlon Brando who have both won the coveted title twice.
Accepting his award, Day-Lewis told the audience in the Dolby Theatre in Hollywood: "I really don't know how any of this happened."
The Lincoln actor – who had previously won the award for roles in My Left Foot in 1990 and 2008 film There Will Be Blood – also paid tribute to his wife, before tearfully thanking his mother.
The other big winners on the night were Anne Hathaway, named best supporting actress for her role in Les Miserables, Jennifer Lawrence who won best actress after starring in Silver Linings Playbook and Ben Affleck's Argo which won best film.
Ang Lee took best director title for Life Of Pi, while chart star Adele triumphed in the best original song category for her Bond theme Skyfall.
She thanked her songwriting partner Paul Epworth for "believing in me all the time, and my man, I love you baby".
Earlier she had performed the song on stage at the event.
On accepting her award, an emotional Hathaway said she hoped for a future in which "the misfortunes of Fantine will only be found in stories and nevermore in real life".
Lawrence, who stumbled on her way to the stage, joked: "You guys are standing up just because you feel bad that I fell over."
Scottish success came from hit cartoon Brave. The film scooped an Oscar for best animated feature film. Directed by Mark Andrews and Brenda Chapman it was voiced by stars including Kelly Macdonald, Robbie Coltrane, Billy Connolly and Craig Ferguson.
There were also wins for Quentin Tarantino's western Django Unchained with Christoph Waltz named best supporting actor, while Tarantino picked up the Oscar for original screenplay.
In a poignant moment, George Clooney introduced a montage of clips dedicated to members of the film industry who died in the last 12 months.