Espargaro, who announced this week that he will retire at the end of the 2024 season, set a time of 1:38.190 on his last lap of the session to leapfrog Ducati's reigning champion Francesco Bagnaia, who was second.
"I don't have 100 pole positions in my career, so even if it's just one Saturday at my home Grand Prix it's amazing, especially fighting with Bagnaia," Espargaro said.
Trackhouse Racing's Raul Fernandez finished third after having a lap time reinstated following the end of qualifying, while Brad Binder of KTM was fourth, ahead of 20-year-old rookie Pedro Acosta.
Championship leader Jorge Martin of Pramac Racing was sixth-quickest after losing his front and crashing out late in the session.
Martin will start on the second row in Sunday's race, giving Bagnaia an opportunity to make up ground on the Spaniard in the overall standings.
"It's always important to start in the front row in Barcelona. Second position is always good," Bagnaia said.
In the first qualifying session, Fabio Di Giannantonio (VR46 Racing) and Raul Fernandez both set lap records to qualify for Q2. Six-time MotoGP champion Marc Marquez of Gresini Racing will start 14th on the grid after failing to make it out of Q1.
