The Catalans have announced that the centre-half is suffering from a back injury and will not feature against los Rojiblancos
The 35-year-old defender informed medical staff of a back problem on the morning of the game at the Ramon Sanchez Pizjuan and has been immediately removed from the team as a precautionary measure.
The Blaugrana captain's last-minute withdrawal adds to the club's concerns in defence, with Jordi Alba and Javier Mascherano both missing the trip to Andalusia through suspension.
"The player has suffered an episode of acute low back pain and is out for the match at the Sanchez Pizjuan," read an official statement from the club on Sunday morning.
This latest setback comes after Puyol, who has made only three league appearances so far this season.
Ibrahim Afellay, who will take the Spaniard's place in the 18-man squad, has himself only just returned to fitness after a long-standing thigh problem.
Barcelona are currently third in La Liga with 54 points from 22 games, three behind both Atletico and Real Madrid, both of whom played a game more.