Puyol, 34, is now tied to Barca until the summer of 2016 after putting pen to paper in the presence of president Sandro Rosell, vice-president Josep Maria Bartomeu and technical director Andoni Zubizarreta.
The defender is Barcelona's captain and has won five La Liga titles, two Copa del Reys and three Champions Leagues since emerging from La Masia.
Puyol will be joined in signing a new contract by both Xavi and Lionel Messi - with Barcelona confirming back in December that all three men had agreed to sign new deals.
However, goalkeeper Victor Valdes confirmed last week that he will leave the club when his contract expires in 2014.
Cesc Fabregas has urged fans not to withdraw their support for goalkeeper Valdes.
"What we have to do is respect his decision and leave it at that because we have to fight for titles," Fabregas told a news conference on Tuesday.
"Football carries on, the club too and the most important thing is the trophies we can win at the end of the season," added the former Arsenal captain.
"We should be worried if he trains poorly and it's the exact opposite. He seems like a youth player at first-team training.
"He has given the club a lot of time to find a solution. He did not think of himself but of the club and the team and what he has decided is proper."
Valdes's announcement has prompted widespread media speculation about who might replace him.
Liverpool's Pepe Reina, who was with Valdes at Barca's youth academy, Manchester United's David De Gea and Atletico Madrid's Thibaut Courtois, on loan from Chelsea, have all been linked with the job.
Valencia's Vicente Guaita, considered the kind of talented ball player Barca would be looking for, is also reportedly in the frame.