Uefa's disciplinary committee met in special session on Thursday and rejected the request.
Real Madrid can take its case to Uefa's appeals body, which could meet next week.
The Spanish champion wanted the pair to be allowed to play in the second round against Liverpool starting on February 25.
The club challenged Uefa to reconsider Article 17.18 of its Champions League regulations that prevents both players being fielded.
The rule says clubs can register up to three newly signed players for the knockout stage before a February 1 deadline, but that only one can have appeared in European competition earlier in the season.
Huntelaar (Ajax) and Diarra (Portsmouth) both played in the Uefa Cup.
Real Madrid's directors have said the club will take its case to the Court ofArbitration for Sport.
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