Both sides have agreed to the 155-pound contest, which will take place on Oct. 30. The UFC has not announced a venue for the event, but it is expected to take place in Abu Dhabi, United Arab Emirates.
Dos Anjos (30-13) and Makhachev (20-1) have gone back and forth on social media this month regarding a potential fight. They were actually scheduled to square off last October, but dos Anjos tested positive for COVID-19. The fight was rebooked in November in Las Vegas, but Makhachev withdrew because of illness.
Dos Anjos, of Rio de Janeiro, told ESPN on Monday he campaigned for the fight to be five rounds but was unsuccessful.
"Islam doesn't want to fight five rounds," dos Anjos told ESPN. "That was my request, but it didn't work out. So, fine. Let's do three then. He's been talking a lot of crap -- let's see who is who now. I'm ready to go. This is a title eliminator [in] my eyes."
Makhachev's manager, Ali Abdelaziz, told ESPN the Dagestani lightweight never had a preference between three or five rounds.
"The UFC offered a three-round fight and Islam said yes," Abdelaziz said. "We don't care about the number of rounds -- it's no problem. The UFC asked for this, and this is what the UFC got. I'm looking at this as a No. 1 contender's match. The winner should fight for the title."
A former champion, dos Anjos is coming off a split decision win against Paul Felder in November. Prior to the win, dos Anjos had competed at welterweight for three years.
Makhachev, a close friend and teammate of retired lightweight champion Khabib Nurmagomedov, is on an eight-fight win streak. He submitted Thiago Moises in the main event of a UFC Fight Night on July 17 in Las Vegas.