Mayfield, who transferred to Oklahoma from Texas Tech, was seeking a waiver on the grounds that he was a walk-on for the Red Raiders and shouldn't be subject to the same transfer rules as a scholarship player.
Instead, he will have to sit out the rest of the season.
After the NCAA denied Mayfield's appeal, Oklahoma officials went back to Texas Tech to see whether the school would certify his immediate eligibility, according to a source, as a last resort. But Texas Tech officials still declined to certify the waiver.
Mayfield, who threw for 2,315 yards after winning the Red Raiders starting job in the preseason as a walk-on true freshman, was the 2013 Big 12 Freshman of the Year.