United dethrone Real Madrid - who held top spot for 11 years - after accumulating a record revenue of 689m euros (£515m) during the 2015-16 term.
The Premier League club saw commercial revenue grow by 100m euros (£71m).
Combined revenue for the top 20 clubs during the 2015-16 season grew 12% to 7.4bn euros (£6.41bn) - a new record.
It is the first time Manchester United have topped the annual Deloitte Football Money League since the 2003-04 season.
Real drop down to third, behind Spanish rivals Barcelona, who remain in second spot.
German giants Bayern Munich move up a position to fourth and Manchester City also climb a spot to fifth - having generated 524.9 euros (£392.6m) - up from 463.5 euros (£352.6m) during the previous season.
It is the first time they have reached the top five of the annual list.
