Omicron cases have been confirmed in at least 15 US states since it was first reported in late November.
There have been only a handful of cases reported in New York.
Mr De Blasio said he was “very concerned.”
He said he expects his new mandate to survive any legal challenges.
A spokesperson for the mayor said it will apply to roughly 184,000 businesses in the city, which has a population of 8.8 million.
Vaccinations are already required for hospital and nursing home workers and for city employees, including teachers, police officers and firefighters.
A vaccination mandate for employees of private and religious schools was announced last week.
Kathryn Wylde, head of the business group Partnership for NYC, said she was “blindsided”.
She told the New York Post: “There’s no forewarning, no discussion, no idea about whether it’s legal or who he expects to enforce it.
“There’s been no consultation. We were blindsided.”
Mr De Blasio leaves office on Jan 1 and will be replaced by another Democrat, Eric Adams.
A spokesman for Mr Adams said: “He will evaluate this mandate and other Covid strategies when he is in office and make determinations based on science, efficacy and the advice of health professionals.”
Republicans rounded on Mr De Blasio.
Lee Zeldin, a US congressman who is seeking the Republican nomination for governor of New York state, called the measures a “job-killing, small business-suppressing mandate.”
He said: “When you dangerously combine a far-left, lame duck politician, who is anti-business, one-dimensional, unaccountable, not bright and has a perpetual ‘I always know best’ attitude,’ you get Bill de Blasio, the Worst Mayor in America.
“I’m hopeful the incoming mayor will roll back these arbitrary mandates.”
Nicole Malliotakis, a Republican US congresswoman representing New York ,said: “He has crushed small business, the economy and quality of life.
“How many more New Yorkers does he want to see move to the free state of Florida?”
But Dr Dave Chokshi, New York City’s health commissioner, said it would “save lives and help prevent unnecessary suffering.”
