The Blaugrana have slammed the move to forbid nationalist flags at the Vicente Calderon ahead of their final contest against Wednesday's Europa League winners
Barcelona have appealed against the banning of Catalan flags at the Copa del Rey final against Sevilla.
Barca had already hit out at the decision not to allow supporters to display the flags for the match at the Vicente Calderon in Madrid on Saturday.
The move had been slammed by vice-president Carles Vilarrubi, who also claimed president Josep Maria Bartomeu may miss the final in protest.
But Madrid governor Concepcion Dancausa said the final "must not be made a scene of political struggle" in announcing the ban.
Barcelona have now confirmed they will appeal to the Madrid courts and believe there has been "an infringement of the fundamental right to freedom of expression".
"FC Barcelona has today [Thursday] filed an administrative appeal to the Madrid Courts for the protection of fundamental rights and a request for a preliminary injunction to suspend the decision that was announced by the delegate of the Community of Madrid," read their statement.
"This resolution prohibits the display of Estelada flags [the name given to flags supporting Catalan independence] at the Copa del Rey final which will take place on Sunday 22 May at the Vicente Calderon stadium, between FC Barcelona and Sevilla.
"The legal basis of the appeal presented by the club is the violation of the fundamental right to freedom of expression and the lack of grounds for the measure because it presupposes a desire to be offensive with the use of the Estelada flags.
"With actions such as these, FC Barcelona defends, and will continue to defend, freedom of expression of its members and supporters."
The Camp Nou club were fined by UEFA over the appearance of Catalan flags in last season's Copa final victory against Athletic Bilbao at Camp Nou.
However, La Liga president Javier Tebas defended the decision of the Madrid courts.
He told AS: "Estelades at that kind of game are a symbol which defends Spain's destruction. Being a game in which public order needs to prevail, these symbols are not allowed.
"I understand and I respect the decision. Estelades can be on the street and also in Liga games, where they are not a problem. But speaking about Spanish Cup final, it's better to ban that symbolism.