The smart gown which she wore as Holly Golightly in the ever-popular film was judged greatest female screen outfit.
The survey, conducted by DVD rental film Lovefilm to mark the release of Sex And The City tomorrow, found Ursula Andress's bikini from Dr No was in the top five.
Hepburn's top gown showed that age was no bar to glamour - her outfit made its appearance almost half a century ago in 1961. One of the three dresses produced for the film sold for more than £400,000 at auction four years ago.
Style queen Hepburn landed a second entry at number six with the white dress and bonnet combo she wore at the races in My Fair Lady.
Runner-up outfit - the floor-length emerald green Jacqueline Durran-designed dress worn by Keira Knightley in Atonement - also had a vintage touch. Although the film dates back only to 2007, the scene in which it features was set in the 1930s.
Marilyn Monroe's cheeky white dress from The Seven Year Itch - which famously blows up as she crosses a subway grating - was placed third.
Andress's beachwear - the briefest outfit on the list - was placed fourth, although running a close second in the skimpy stakes is Raquel Welch's leather and fur bikini from One Million Years BC.
Style icon Sarah Jessica Parker is ranked 10th for the Vivienne Westwood wedding dress for the first Sex And The City movie.
Lovefilm editor Helen Cowley said: "Audrey Hepburn has truly made that little black dress a fashion staple which has stood the test of time despite competition from some of the most stylish females around.
"As Sarah Jessica Parker and co-stars prepare to provide the next fashion fix for women everywhere, it'll be interesting to see if their latest threads can make the same impact."
Sex And The City 2 has its UK premiere tonight in London