Mazembe, who were seeking an unprecedented third successive continental title, were found guilty of fraud by the competition's organising committee at their regular mid-season meeting in Cairo.
Mazembe used Janvier Bokungu, who CAF found was contracted with another club but used by Mazembe in both legs of their second round tie against Simba SC of Tanzania in April.
Bokungu, 22, was suspended earlier this month by African football's governing body despite Mazembe's assertion he had terminated his contract in Tunisia to re-join his old side.
Mazembe's owner, Moise Katumbi, denied any wrongdoing and said they would appeal against CAF's decision.
"We are very surprised to see that Mazembe has been disqualified, we know our administration (of the club) is very clean," he told Reuters by telephone, adding that Mazembe's fans were shocked at the news.
A CAF statement said Simba would be reinstated into the competition and would play a one-off match with Morocco's Wydad Casablanca, who lost on aggregate to Mazembe in the third round last weekend.
"Given that we received your letter asking for the postponement of league matches on the same day you flew to Brazil, we consider this a deliberate attempt to make us accept it as a 'fait accompli'," said a statement from the FECOFA, the controlling body.
However, Katumbi told Reuters the team had received permission to travel to Brazil and there were "no problems" with the Congolese football authorities.
