The tribunal rejected an appeal by Portuguesa, which had lost four points for using a suspended player in the tournament's final round.
Without the points, Portuguesa drops below Fluminense in the 20-team standings and has to replace the traditional Rio de Janeiro club in the second division next year.
Newly acquired Toronto FC striker Gilberto was loaned to Portuguesa this season by Internacional. He did his part to keep them up, scoring 14 goals in 24 games. He did not play in the controversial final round game that involved the disputed player.
The ordeal further tarnishes the image of football in Brazil, which recently made headlines for fan violence and the deaths of workers at World Cup stadiums.
Fluminense, a four-time national champion, had become the first defending champion to be relegated in the Brazilian league.