Messi limped off after less than half-an-hour and could be absent for up to two months after scans revealed the full extent of the problem in his left hamstring.
The Argentinian playmaker will miss the upcoming international friendlies against Ecuador and Bosnia and Herzegovina.
Barcelona have already qualified from the group stages of the UEFA Champions League, but Messi will be absent for a number of La Liga matches.
The world player of the year has been hampered by hamstring issues and also damaged his left one earlier in the season and then the right one at the end of September.
A club statement read: "According to the club's medical services, the tests taken on Monday morning by Leo Messi have confirmed that the player has suffered a torn muscle in the femoral biceps of his left leg.
"The approximate time he will be out is between six and eight weeks.
"The first part of his recovery will take place in Barcelona and the second, in Buenos Aires."
Barca team-mate Cesc Fabregas has pulled out of the Spain squad after he also sustained a knee injury against Betis.
Fabregas netted twice before being withdrawn and Spain boss Vicente del Bosque has decided to rest him for friendlies with Equatorial Guinea and South Africa.
Defender Marc Bartra, another Barcelona player, has been drafted in to replace Fabregas and the 22-year-old will be hoping for his first senior cap.