The track, which also features rapper T-Pain, spent ten weeks at No. 1 on the Billboard Hot 100 from January 2008 and has notched up over 5 million downloads.
Lady GaGa's 'Just Dance' is the second most downloaded song, while her follow-up 'Poker Face' is in seventh place.
The Top 10 shows that download figures have increased substantially over the last few years, with tracks from Black Eyed Peas and Taylor Swift selling more than others managed in a decade.
The Top 10 most downloaded songs in the US of the 2000s (total sales in brackets):
1. Flo Rida ft. T-Pain: 'Low' (5,214,000)
2. Lady GaGa ft. Colby O'Donis: 'Just Dance' (4,690,000)
3. Jason Mraz: 'I'm Yours' (4,619,000)
4. Timbaland ft. One Republic: 'Apologise' (4,439,000)
5. Black Eyed Peas: 'Boom Boom Pow' (4,349,000)
6. Soulja Boy: 'Crank That' (4,315,000)
7. Lady GaGa: 'Poker Face' (4,200,000)
8. Coldplay: 'Viva La Vida' (4,140,000)
9. Taylor Swift: 'Love Story' (4,005,000)
10. Katy Perry: 'Hot N Cold' (3,945,000)