A crowd can be heard chanting "Vucicu, pederu" ("Vucic, faggot") in the clip, which was intended to highlight insults directed at the premier from his rivals ahead of the first round of the presidential election next month.
But the video sparked anger from gay rights groups and Serbia's electronic media watchdog said it was in breach of regulations.
"An advertising message cannot contain statements or visual presentations which can be considered offensive," the monitor said in a statement, giving media a 24-hour deadline to cease broadcasting the clip.
Vucic, who heads the Serbian Progressive Party (SNS) and became premier in 2014, is leading opinion polls ahead of the first round of the presidential election on April 2.
A former ultra-nationalist who has restyled himself as a pro-European reformist, 47-year-old Vucic is expected to remain Serbia's most powerful politician if he wins, although the presidential role is largely ceremonial.
More than 80 percent of Serbia's 7.1 million-strong population are Orthodox Christian and society remains deeply traditional.
Homophobia is widespread. At Belgrade's Gay Pride march in 2010, hardline nationalists attacked participants and clashed with police, wounding 150 people and prompting officials to ban the parade for three years.
The event has passed off peacefully for the last two years - though under a heavy police presence - with organisers reporting a significant decrease in threats from far-right groups.
Last year Ana Brnabic became the first openly gay member of the Serbian cabinet when she was named minister for state administration.
