Franco Frattini, president of the Italian Olympic Committee (CONI) tribunal, told reporters CONI had voted 3-2 not to allow any additions to the championship which started two weeks ago.
Serie B usually has 22 teams but three of those who should have played this season were barred for financial reasons.
Bari and Cesena, both former Serie A clubs, were declared bankrupt while Avellino were refused a licence due to a lack of financial guarantees, leading to a close season of chaos in the lower reaches of Italian football.
The Italian football federation (FIGC) began drawing up plans for a playoff competition to find three replacements but in August accepted a request from the Serie B league to go ahead with 19 clubs.
However, six clubs then appealed to CONI -- Virtus Entella, Novara, Pro Vercelli and Ternana, who were relegated from Serie B last season, plus Siena and Catania, who reached the Serie C promotion playoffs.
The CONI tribunal had the three options of rejecting the appeal, allowing all six to take part in Serie B or organising a playoff competition to determine three extra participants.
Mauro Balata, president of Serie B, welcomed the ruling and said it could usher in long-term change.
"Now, Serie B can continue with a reduction in the number of teams which could signal the change of a system which has become unsustainable and incapable of reforming itself," he said.
Tuesday's decision means the third tier Serie C championship, which features 60 teams and has twice been postponed due to uncertainty over who will take part, can finally draw up its fixture list and get underway.
(Writing by Brian Homewood; Editing by Ken Ferris)
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