Landis, 31, failed a drug test after the Tour's 17th stage, when he was found to have used a banned synthetic testosterone.
After his victory, he was stripped of his title, and banned for two years by the International Cycling Union.
In May, Landis testified before a three-member panel that he had not used drugs and cited incompetence at the French laboratory where his urine sample was tested.
"This ruling is a blow to athletes and cyclists everywhere," Landis said in a statement. "I am innocent, and we proved I am innocent."
If Landis does not appeal Thursday's ruling, he will be the first person in the tour's 105-year history to lose the title because of a doping offense.