In the main event at a sold-out 15,000 RAC Arena, heavy favourite Lomachenko defied the rowdy home crowd and dominated throughout.
Lomachenko left the Australian bloodied and sealed the 135-pound belt in the 11th round after dropping Kambosos several times before the fight was stopped by the referee.
It was the first IBF lightweight belt in the glittering career of former WBA and WBC lightweight champion Lomachenko, who has also won world titles in featherweight and super featherweight. He is also a two-time Olympic gold medallist.
"I'm again world champion. I want to come back home and spend time with my family," said 36-year-old Lomachenko, who remained coy about his future.
Kambosos, a former champion, was unable to reproduce the form from his famous upset of Teofimo Lopez in 2021 at Madison Square Garden.
His pre-fight taunts of sending the 36-year-old Lomachenko into retirement fell flat.
"He's one of the best of all time. We tried our best, but he's a true champion," said Kambosos, who was defeated for the third time in 24 fights.
"I gave it my all. I gave it everything in my training camp."
Both fighters had a point to prove after losing to American Devin Haney in recent years.
Kambosos, who made his ring walk to the Eminem song 'Till I Collapse', needed to rely on his formidable striking and size advantage.
He came out aggressively and attacked the body of Lomachenko, who is sometimes an unusually slow starter.
Lomachenko got back into the contest through trademark lightning foot speed and counter-punching.
The brash Kambosos was unruffled and taunted Lomachenko after trading a flurry of heavy blows to end the third round.
Undercard controversy
The partisan crowd was right behind the Australian, but southpaw Lomachenko stepped up the intensity in the middle rounds and landed several stinging left-handed jabs that rattled his opponent.
Lomachenko strengthened his grip on the fight in the latter rounds, with several hard left-handed blows leaving Kambosos with blood flowing from his right eye.
Lomachenko thought he had knocked Kambosos down in the 11th, though it was ruled a slip, but then a vicious body shot did drop the Australian for an eight count.
Several more ferocious blows prompted the referee to stop the fight after Kambosos sank to his knee in his own corner, moments before the towel came in from Kambosos's trainer.
The showdown had been a long time coming, with plans falling through in 2019 before a fight three years later was scuppered when Lomachenko returned home to his family because of Russia's invasion of Ukraine.
In the two world title fights on the undercard, Mexican Pedro Guevara beat Australia's Andrew Moloney with a contentious split decision to claim the interim WBC super flyweight belt.
An aggrieved Moloney, a former champion who battled through a torn bicep, announced his retirement immediately after the fight.
"I'm done with this sport. That's the last you'll see of me," the 33-year-old said.
Australian Cherneka Johnson stripped England's Nina Hughes of her women's WBA bantamweight belt after winning the 10-round battle in a majority decision.
There was confusion after the ring announcer initially awarded the victory to Hughes after misreading the scores.
