Barcelona will go to the Court of Arbitration for Sport after Uefa dismissed the club's appeal against a £29,000 fine imposed for fans showing support for Catalan independence.
Fans chanted and waved Catalan flags at Barcelona's Champions League home win over Bayer Leverkusen in September.
Uefa charged Barca with "transmitting messages" of a "political nature".
They were fined £22,000 for a similar offence in last July's Champions League final win against Juventus.
The club has long been an outlet for fans who support the independence of Catalonia from Spain with supporters displaying flags, known as "Esteladas".
Supporters were criticised by politicians from Spain's ruling People's Party after they whistled the national anthem at the Copa del Rey final at the end of May.