Portugal took an early two-goal lead thanks to Liverpool forward Diogo Jota’s brace but Serbia pegged back the reigning European champions with two second half goals of their own to draw level.
Ronaldo seemed to have snatched all three points for Portugal deep into second half stoppage time when his effort crossed the line just before a defender hooked the ball away.
Instead, referee Makkelie waved play on and Ronaldo was booked for taking his protests too far on a day when VAR was not in operation to intervene.
"As per FIFA's policies, all I can say is that I apologised to the national coach, Fernando Santos, and to the Portuguese team for what happened," Makkelie told Portuguese publication A Bola.
"As a referee team, we always work hard to make good decisions. When we are in the news in this manner, it doesn't make us happy at all."
Portugal are second in Group A behind leaders Serbia and they travel to Luxembourg in their next outing on Tuesday.
