The encampment must be closed, and all tents removed, by July 17, city officials said.
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The encampment began to form last month.
Organizers say the goal was to shine a light on homelessness in the city, and support the Black Lives Matter movement.
The closure announcement came after representatives from the city's offices of health and human and services and homeless services held a remote meeting on Friday morning.
Homeless protest forms encampment on Ben Franklin Parkway
Mayor Jim Kenney's office said that the order came after weeks of negotiations to respond to "ever-shifting demands" by representatives and residents of the encampment.
Kenney's office said officials are increasingly concerned about public health and safety.
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However, protesters said Friday they're not budging.
"They think we're just out here doing whatever we want. Guess what city, so are you," encampment nurse Jonnell Johnson Flowers said.
"We may not be sitting on Society Hill but that don't make us dumb," Leonard Flowers said.
The founder of the movement, Leonard Flowers, and his wife said the announcement from the city was a slap in the face.
"You take it upon yourself and make a decision about our lives with no notice, no backup plan - it's not okay," Jonnell Johnson Flowers said.
The camp has been in place for more than a month as people fight for permanent housing. The city says it tried to reach an agreement peacefully but when outreach workers entered the camp this week they say some living here got violent.
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"The city say they came out and someone got violent with them. Nobody from the city did come out," Leonard Flowers said.
Officials admit there's no full plan ironed out if people in the encampment refuse to move. But they say there are beds in shelters to place people or some can seek treatment in the COVID-19 center. Officials say they will be at the encampment to work with people to figure out their next move.
"With protests comes a fight," Jonnell Johnson Flowers exclaimed.
The protesters said they won't be gone by the city's deadline. Officials did not want to comment on what happens if people refuse to leave or say if police would be called in.
The city did mention possibly moving this encampment to another location but haven't figured out where.