In a strongly-worded statement released on Monday, the UCI said the allegations made by Armstrong's former team mate Tyler Hamilton on the US television version of "60 Minutes" on Sunday were "completely unfounded".
"The International Cycling Union categorically rejects the allegations made by Mr Tyler Hamilton, who claims that Lance Armstrong tested positive for EPO during the 2001 Tour of Switzerland and had the results covered up...," the statement said.
During the interview, Hamilton confessed to cheating himself but insisted he was not alone and painted a sordid picture of a doping culture in the sport.
The former Olympic champion, who handed back his gold medal last week after his admission, said doping was widespread and he pointed the finger at seven-times Tour de France champion Armstrong, saying the American not only cheated but also escaped punishment after being caught.
The UCI categorically denied the accusations made by Hamilton, accusing him of trying to discredit the sport.
"The UCI is deeply shocked by the seriousness of the allegations...and wishes to state once again that it has never altered or hidden the results of a positive test," the UCI said.
"The allegations of Mr Tyler Hamilton are completely unfounded. The UCI can only express its indignation at this latest attempt to damage the image of cycling by a cyclist who has not hesitated to abuse the trust of all followers of cycling on several occasions in the past.
"At no time did he see fit to inform the UCI of the events he claims to have witnessed 10 years ago, and which he is now using in his attempt to harm the UCI.
"The UCI can only confirm that Lance Armstrong has never been notified of a positive test result by any anti-doping laboratory."
Armstrong has repeatedly had to fend off accusations that he cheated despite always maintaining his innocence and never failing a dope test.
Two former team-mates, Floyd Landis and now Hamilton, have said he tested positive for EPO (erythropoietin) during the 2001 Tour of Switzerland but escaped punishment after the UCI intervened on his behalf.
The UCI commenced legal action against Landis this month and hinted that it could do the same with Hamilton.
"The UCI will continue to defend its honour and credibility by all means available, and reserves the right to take any measures it deems necessary against Mr Hamilton or any other person," the ruling body said.
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