United were valued at $3.69bn (£2.86bn) and returned to the top of the annual list for the first time in five years.
Spanish club Barcelona were second with a worth of $3.64bn (£2.82bn) as Real $3.58bn (£2.77bn) went down to third.
Six English teams were in the top 10, with Manchester City, Arsenal, Chelsea, Liverpool and Tottenham joining United.
German champions Bayern Munich were fourth, while Italian title winners Juventus were ninth.
Real, who won the Champions League and Spanish title, had been top for the past four years but saw their value drop by 2% as United's went up by 11%.
"Manchester United's return to the top spot is a testament to their powerful brand and marketing acumen," Forbes Media assistant managing editor Mike Ozanian said in a statement.
Leicester, who won the English Premier League in 2015-16, were 19th and West Ham 15th.
Forbes' top 20 most valuable football teams
- 1. Manchester United $3.69bn (£2.86bn)
- 2. Barcelona $3.64bn (£2.82bn)
- 3. Real Madrid $3.58bn (£2.77bn)
- 4. Bayern Munich $2.71bn (£2.1bn)
- 5. Manchester City $2.08bn (£1.61bn)
- 6. Arsenal $1.93bn (£1.5bn)
- 7. Chelsea $1.85bn (£1.43bn)
- 8. Liverpool $1.49bn (£1.15bn)
- 9. Juventus $1.26bn (£976m)
- 10. Tottenham $1.06bn (£821m)
- 11. Paris St-Germain $841m (£652m)
- 12. Borussia Dortmund $808m (£626m)
- 13. AC Milan $802m (£621m)
- 14. Atletico Madrid $732m (£567m)
- 15. West Ham $634m (£491m)
- 16. Schalke 04 $629m (£487m)
- 17. Roma $569m (£441m)
- 18. Inter Milan $537m (£416m)
- 19. Leicester City $413m (£320m)
- 20. Napoli $379m (£294m)