The flamboyant star - real name Stefani Germanotta - moved up four positions from last year to knock Winfrey, 2010's chart-topper, down to two.
Gaga's ranking takes into account her earnings over the past 12 months - an estimated $90m (£55.6m).
It also cites her 32 million Facebook fans and 10 million Twitter followers.
"Lady Gaga beat Oprah this year because of her social media power," said Forbes editor Dorothy Pomerantz said.
"She's the best example of how celebrities will need to manage their careers in the coming years."
Canadian pop star Justin Bieber makes his first appearance on the list in third place, thanks in part to his estimated earnings of $53m (£32.7m).
Irish rockers U2 come fourth in the countdown with estimated pre-tax income between 1 May 2010 and 1 May 2011 of $195m (£120.5m).
Sir Elton John is the highest ranked Briton on the list, which measures celebrity "power" by entertainment-related earnings, media visibility and social media popularity.
He is joined in the Top 10 by Simon Cowell, whose role as music producer and X Factor judge earns him ninth place in the annual countdown.
Lady Gaga's latest achievement coincides with the imminent release of her latest record, Born This Way.
All 14 tracks from the album were streamed online in Europe this week ahead of its official release on Monday.