The annual review of soccer finance by Deloitte showed that Madrid generated 438.6 million euros ($539.1 million) in the year ending last June 30, an increase of 20 percent as the club retained the top spot for the sixth straight year.
Barcelona, which beat Madrid for the Spanish title last year and won the 2009 FIFA Club World Cup, saw a more modest revenue increase of just under 10 percent to 398.1 million euros ($489.3 million).
The rivals' supremacy is helped by the uneven distribution of broadcasting rights from Spanish competitions.
The top six remained unchanged from last year's rankings, with Manchester United third, followed by Bayern Munich, Arsenal and Chelsea.
The biggest mover in the top 20 was Manchester City, with the Premier League's wealthiest club climbing nine places to 11th with a 44 percent revenue increase to a club record 125 million pounds ($187.8 million).
Premier League rival Liverpool dropped one place to eighth after finishing seventh and failing to qualify for the Champions League.
"All bar three of the top 20 clubs achieved revenue growth during 2009-10 demonstrating the continued resilience of football's top clubs as the full impact of the global economic downturn took hold," Dan Jones of the Deloitte sports business group said. "The game's top clubs have proved themselves well-placed to meet these economic challenges given their large and loyal supporter bases, ability to drive broadcast audiences, and continuing attraction to corporate partners."
The combined income generated by the top 20 clubs was up 8 percent on the previous year. Deloitte said that 44 percent of revenue came from broadcast rights.
Fourteen of the top 20 clubs participated in the Champions League and the remainder competed in the Europa League.