Levy picked up 40% of the votes in the LoveFilm poll of 1,200 people, beating Chevy Chase as Clark Griswold from National Lampoon's Vacation into second place.
Levy's character was famous for making his son Jim feel extremely awkward while giving advice on life and sex.
Homer Simpson (voiced by Dan Castellaneta) was voted in third place from The Simpsons Movie, followed by Robin Williams as Mrs Doubtfire in fourth.
Other entries in the top ten include Robert De Niro as former CIA agent Jack Byrnes in Meet the Parents and Jim Carrey as Fletcher Reede in Liar, Liar.
LoveFilm editor Helen Cowley said: "On the day we show our appreciation to the father-figures in our lives it seemed only fitting that we remember those hilarious and often cringeworthy dads in film who make us laugh at the expense and embarrassment of their kids."
The top ten in full:
1. Eugene Levy – American Pie (40%)
2. Chevy Chase – National Lampoon's Vacation (20%)
3. Homer Simpson – The Simpsons Movie (13%)
4. Robin Williams – Mrs Doubtfire (8%)
5. Robert De Niro – Meet the Parents (7%)
6. Rick Moranis – Honey, I Shrunk the Kids (4%)
7. Steve Martin – Parenthood (3%)
8. Jim Carrey – Liar Liar (3%)
9. Eddie Murphy – Daddy Day Care (1%)
10. Shrek – Shrek Forever After (1%)
It has been confirmed that Eugene Levy will return for the fourth American Pie movie, titled American Reunion, to be released on April 6, 2012.
Watch a scene featuring Eugene Levy as Jim's dad from American Pie 2 below: