The 29-year-old's triumph on his home circuit meant standings leader Marc Marquez, who finished second by 1.740 seconds, cannot seal a seventh world title next weekend at Misano.
Ducati rider Marc Marquez leads the overall standings on 487 points, with Alex trailing by 182 after his second career victory and the second of this season.
Enea Bastianini came in a further 5.562 seconds behind on his KTM to finish third.
"I'm very happy, very pleased, but to be honest, I still had a thorn in my side from yesterday," said Alex Marquez, who crashed from the lead during the Sprint on his Gresini Ducati.
"Today I had to pull that thorn out - maybe without that mistake yesterday, I would have had it today, so maybe it served for something.
"It was a race of self-control. I had more, but I didn't want to destroy the tyres."
Even though he will have to wait longer to complete his title charge, Marc Marquez was content with the day's racing.
"I gave everything. I had the perfect race. I led on the first laps," he said.
"On the last laps, I was on the limit; it was hard for me.
"I'm happy for Alex, for Ducati and for me, because on a difficult circuit I came second."
On Saturday, polesitter Alex Marquez watched in frustration as his brother claimed victory in the Sprint after his surprise crash four laps from the end.
This time, the 32-year-old nosed ahead of Alex into the first turn of the race from third on the grid.
Alex battled back to retake the lead on turn one of lap four before shutting out his brother on the second turn to open up a lead which he never relinquished.
Alex Marquez held off his brother well, growing out the gap to 0.6 seconds.
Every rider used medium/medium tyres apart from Pedro Acosta on medium/soft, and perhaps that began to tell as he fell a long way behind third-place Bastianini by the end.
The two Marquez brothers outstripped their competition by far, with Marc Marquez over four seconds ahead of third-place Bastianini during the final lap.
The brothers celebrated together with abandon in front of delighted fans at a packed Circuit de Barcelona in Montmelo.
At the earliest, Marc Marquez can now seal the title at the Japanese MotoGP on September 28th.
Amid a crisis of confidence, Francesco Bagnaia started 21st on the grid, after his worst qualifying performance since 2022, but produced an encouraging display to finish seventh.
