United had held first place since business analyst Forbes started compiling the list in 2004, but have been overtaken by Real in the latest rankings.
Barcelona, the current Spanish league leaders, are in third place, while United's Premier League rivals Arsenal are fourth ahead of newly crowned German champions Bayern Munich.
Real Madrid are valued at £2.15 billion ($3.3 billion), making the Spanish club the most valuable sports franchise in the world, and United at £2.07 billion.
Forbes said in a statement: "This is the first time since Forbes began valuing soccer teams in 2004 that Manchester United is not No.1 on the list.
"Real Madrid has been consistently delivering the highest revenues of any soccer team in the world, despite an intense economic meltdown in Spain.
"With superstars like Cristiano Ronaldo on the pitch, and coach Jose Mourinho on the bench, Real Madrid has thrived under the leadership of billionaire president Florentino Perez.
"According to this year's soccer valuations, the top 20 teams are worth an average of $968 million (£634 million), an increase of 26 percent over last year."
Paris Saint Germain's David Beckham still tops the list of highest-earning footballers with earnings of £33 million, £29 million of which came from endorsements.
Ronaldo is second with earnings of £28.8 million and Barcelona's Lionel Messi in third place with a total of £26 million last year from salary and endorsements.
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