Messi came off the bench to help Barca beat Leganes in LaLiga at the weekend, but Ernesto Valverde left his captain out entirely at the Ramon Sanchez-Pizjuan on Thursday (AEDT), handing a debut to shock signing Kevin-Prince Boateng in attack instead.
Luis Suarez and Philippe Coutinho came on as substitutes but, if Barca are to win the Copa del Rey for a fifth year in a row, they will have to overturn a first-leg deficit as they did in the last round against Levante.
Sarabia struck to capitalise on a period of Sevilla dominance with Ben Yedder adding a late second, Pablo Machin's side bouncing back from a run of three straight losses in all competitions to stun the holder, although they lost talismanic winger Jesus Navas to injury.
Sevilla thought they had been awarded a penalty in the first half when Arturo Vidal blocked a cross with his upper arm, but a VAR review opted not to give a spot-kick.
Ben Yedder should have given Sevilla the lead in the 36th minute, brilliantly beating Gerard Pique during a dazzling solo run but slicing his shot badly wide of the post.
Barca missed an even better chance before the break, Malcom running on to a sublime Arthur pass to round Juan Soriano but hitting his finish into the side-netting.
Quincy Promes and Ibrahim Amadou wasted good opportunities after the restart as Sevilla continued to impress, the opening goal deservedly arriving in the 58th minute.
Promes raided down Sevilla's left wing and floated a cross into the Barca box, Sarabia meeting the ball with an excellent first-time finish that gave Jasper Cillessen no chance.
And Sevilla gave itself a further cushion ahead of the return fixture when it scored what might prove to be a decisive second goal in the 76th minute.
Andre Silva and Ever Banega combined to tee up Ben Yedder at the back post, the striker using his celebration to show a message of support to missing Cardiff City striker Emiliano Sala.