The film starred Joaquin Phoenix as US country legend Cash and Reese Witherspoon as his wife and stage partner June Carter Cash, for which she won the Best Actress Oscar in 2006. The movie was nominated for five Oscars in total.
Walk The Line beat rapper Eminem's film 8 Mile, Mozart's life story Amadeus and another Oscar winner, Ray, starring Jamie Foxx as Ray Charles, to the top spot.
The most recent film in the top 10 was Joy Division biopic Control, which debuted at Cannes Film Festival and came out in the UK earlier this month. In fifth place was The Doors, featuring Val Kilmer as doomed star Jim Morrison.
More than 2,000 film fans were polled by online DVD firm Lovefilm. A spokeswoman for the company commented: "Film and music make a great partnership and have resulted in some of the best acting performances in film.
"This poll showcases the very best the genre has to offer - whether it be a portrayal of a foul-mouthed rapper or a classical composer."