The Argentina star was suspended for a red card in the Supercopa de Espana final defeat to Athletic Bilbao, in which he was sent off for striking Asier Villalibre after a VAR (video assistant referee) review.
It was the first time in 753 appearances for the club in all competitions that Messi had been dismissed.
Barca appealed against the suspension but confirmed on Saturday (AEDT) that its efforts had been in vain, meaning Messi will not be available for the game at Estadio Martinez Valero.
The six-time Ballon d'Or winner had already served one match of his ban, having been absent from Friday's (AEDT) Copa del Rey win at Cornella.
Ousmane Dembele and Martin Braithwaite scored in extra-time after Barca missed two penalties against the third-tier side.
Messi will be free to return in next Thursday's (AEDT) Copa del Rey Round of 16 clash against Rayo Vallecano, with another meeting with Athletic, this time in LaLiga, to follow on 1 February (AEDT).