The Argentinian moved up to 86 goals in 2012 but was only given the go-ahead to play against Betis before the game after injuring his knee in the midweek Champions League game against Benfica.
He beats the German legend's record set in 1972, and now has 23 in the league this season alone, six more than Radamel Falcao who hit five goals for Atletico Madrid in their 6-0 Deportivo La Coruna.
Aterwards the unassuming Barcelona superstar was happier to highlight the win more than the record as his team stayed six points clear at the top.
"I've said it a lot, it's nice to beat records but the win for the team is what is important and the points difference at the top.
"My goals are to win titles with the team, the Spanish league, Spanish Cup or Champions League is more important than any personal records," he said.
Barca's Sporting Director, Andoni Zubizarreta, was more forthcoming.
"I think his records will be valued more in the future, it's one more for now but he makes it seem so easy it's hard to measure his talent. It's a gift to see him play football but I think he'll say the same, its a team game and he needs his team mates to help him," he said.
Barca coach Tito Vilanova looked forward to more to come.
"It seems impossible to score so many goals, let's hope he can add to the record in the games to come," said the coach.
"He is still young and gives us so much, even tonight he helped us out defensively when we needed it, he has so many more qualities than just his goals."
It did not take long for Messi to equal Mueller's record with his side's first real attack on 16 minutes, making a diagonal run across the Betis defence before hitting a left footed shot into the right hand corner.
His history-making moment came nine minutes later when he struck from almost an identical position after good play from Adriano and Andres Iniesta.
Betis had started and finished the better side in Seville, Salva Sevilla had a header well saved by Victor Valdes on three minutes and they hit the woodwork on three occasions in the second-half.
Adrian in the Betis goal then made an acrobatic save to deny Xavi Hernandez, and the home side appeared out of the game until substitute Alvaro Vadillo played a clever pass to Ruben Castro who finished coolly to reduce Barca's lead before the interval.
Valdes saved Barca six minutes into the second period, plucking a left-wing volley from Sevilla from under the crossbar.
The game continued from end-to-end at a frantic pace without many clear cut chances until Barca's Javier Mascherano struck his own post whilst trying to clear a corner with 20 minutes remaining.
Jorge Molina then hit a post with a fierce shot while at the other end Adrian blocked well from Pedro Rodriguez before Jordi Alba hit the bar when he should have scored.
Barca were in danger of conceding until the end but held on for a vital three points where rivals Real Madrid lost 1-0 only two weeks ago.
The win keeps Barca six points ahead of Atletico Madrid who had earlier thrashed Deportivo La Coruna 6-0.
Falcao became the first La Liga player in a decade to score five to keep the pressure on Barca.
He had already hit a double along with a Diego Costa header to put Atletico out of sight by half-time, his second smashed on the half-volley direct from a throw-in.
He completed his hat-trick with a second-half penalty before hitting two more to make him the first player to hit five in a La Liga game since Fernando Morientes for Real Madrid against Las Palmas in 2002.
It was Atletico's eighth straight home win of the campaign.
A hapless looking Deportivo side remain bottom of the table on 11 points, level with Espanyol.
Also on Sunday, Levante thrashed Mallorca 4-0 with goals from Obafemi Martins, David Navarro, Ruben Garcia and Vicente Iborra to move into sixth position.
A first-half header from Aritz Aduriz, his ninth of the season, was enough for Athletic to edge a competitive encounter with Celta Vigo.
On Saturday Real Madrid kept in touch with a 3-2 win at Valladolid, but remain five points behind Atletico in third.