The unanimous decision means Portuguesa will drop into Serie B next year even though they ended the season two points and five places ahead of Fluminense, who had been the first team to be relegated a year after winning the league title.
Portuguesa, a small club from Sao Paulo, made the mistake in the final match of the season a week last Sunday.
"We're very sad, but we expected it," full-back Brayan said.
"We can appeal and see what we can do. But this was no surprise. We have to accept it, no matter what division we're in we'll sweat to do our best."
They were punished for introducing Heverton in the 77th minute of a scoreless draw against Gremio. The midfielder had not sat out the second game of his two-game suspension.
Portuguesa said during Monday's hearing that their lawyer had not informed them of the outstanding suspension and said it would be overly harsh to relegate them for fielding a player who played 13 minutes and did not affect the outcome of the game.
But judges said ignorance was no defence.
"It's no excuse for them to say they didn't know," said judge Paulo Valed Perry. "There was a clear mistake by Portuguesa."
It was not the first time Fluminense, traditionally the club of Rio de Janeiro's white elites, have been saved from the second division by controversial off-the-field decisions.
In 1999, the team won Serie C and were scheduled to play in Serie B a year later. However, an unanticipated reform of the league system saw them lifted straight into the first division.
In a later decision on Monday, the court also docked four points from Flamengo.
The Rio club played former Arsenal full-back Andre Santos against Cruzeiro when he should have sat out a match for suspension.
Flamengo argued the suspension was picked up in a Cup tie and that the punishment should not apply to a league game.
The decision means that Flamengo end the season in 16th place, just one point above the revised relegation zone.