The Spanish side's value fell 5% to $3.26bn (£2.14bn) from 2014 but they had the highest revenue of any sports team in the world at $746m (£489m).
Fellow La Liga giants Barcelona, worth $3.16bn (£2.07bn), are second while Manchester United sit third with $3.10bn (£2.03bn), a 10% increase.
United are one of eight Premier League teams in the top 20.
Manchester City are fifth behind German champions Bayern Munich, while new Premier League champions Chelsea are sixth.
Arsenal are seventh and Liverpool eighth, with Serie A sides Juventus and AC Milan completing the top 10.
Rising television and shirt sponsorship revenue sees the average value of the top 20 clubs at $1.16bn (£761m), an increase of 84% from five years ago.
United have the second biggest revenue at $703m (£461m) despite finishing seventh in the Premier League last season and missing out on Champions League qualification.
The Red Devils signed a £750m, 10-year kit deal with Adidas that starts next season.
City's value has risen by 59% over the past year which saw them win the Premier League title, while Chelsea's value has increased by 58%.
Tottenham (13th), Newcastle (18th) and West Ham United (19th) are the other Premier League teams in the top 20, which Forbes credits to both a rise in the British pound relative to the euro and US dollar and a lucrative £5.14bn TV deal starting from the 2016-17 season.
Real Madrid have topped Forbes' annual list of the world's most valuable teams for the third consecutive year.