The Italian league imposed the sanction last week after a section of Juve fans repeatedly racially abused Inter Milan striker Mario Balotelli in a 1-1 draw in Turin earlier this month.
The Italian soccer federation, which is battling to stamp out a racism problem among fans, said in a statement on Tuesday that Juve's appeal had been rejected and that Sunday's game would have no spectators.
The club were criticised in the media for trying to overturn the ban amid pressure from some supporters, who said Juve were being singled out when incidents of racism at other clubs were only punished with fines.
The Turin club were demoted to Serie B in 2006 for their role in a match-fixing scandal.
A group of Juve fans have slammed club bosses and coach Claudio Ranieri in a number of recent protests after the side slipped back to third in Serie A and were dumped out of the Italian Cup semi-final by Lazio.
Chief Executive Jean Claude Blanc has given his backing to Ranieri but media speculation about his possible departure has been rife with Italy coach and former Juve boss Marcello Lippi linked with a return after the 2010 World Cup.
