The 32-year-old World Cup winning central defender has failed to recover from a left knee injury, which has kept him on the sidelines for the past several weeks and he felt pain in the offending knee after taking part in two training sessions at the weekend.
His absence means that Barca will be without either of their first choice central defenders as Gerard Pique is suspended.
Barca goalkeeper Pep Guardiola will have to choose his pairing from three players: French left back Eric Abidal, Gabriel Milito, who has rarely played this season, and defensive midfielder Sergi Busquets.
Puyol ruled out of Arsenal clash
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