Organisers of the "People's Vote" campaign said they wanted to ascertain whether new Brexit terms by Johnson were good for the UK, the BBC reported.
Protesters were headed to Westminster as MPs debate the new deal in the Commons.
The march, which began at midday on Park Lane, will end in Parliament Square.
Ali Lothian, 60, and Mettje Hunneman, 49, travelled from Dundee and Edinburgh, respectively, to join the protest.
Ali told the BBC she felt it was the last chance to show how strongly she felt about having another vote. "It's a big commitment. It's a whole weekend. But I regretted not coming last time. This time it was a no-brainer," she said.
Mettje said that Parliament's sitting on Saturday made it "a momentous day". "I would not feel comfortable sitting at home. I've got pals who have got a gig tonight but I just couldn't be there," Mettje said.
Millie Bishop-Morris, 17, made the journey from Plymouth with her mother and boyfriend. "I think it's important that young people should be angry about this as well," she said.
"I think Brexit has gone completely the wrong way. I want to be optimistic, but I'm preparing myself for the worst," she said.
One group of protesters were seen pulling a float depicting top aide Dominic Cummings using Johnson as a puppet. With "Demonic Cummings" splashed across its forehead, the figure on the float appears to be wearing a Nazi uniform, including an armband that reads Get Brexit Done, and has a Union Jack moustache.
As of Saturday morning, more than 500,000 pounds has been donated to support the protest, and cross-party politic are calling on people to get involved.
People's Vote organisers are also asking people to sign a letter to Johnson, EU leaders, MPs, and MEPs, asking them to allow, "the chance to check whether we want to proceed with Brexit".
In an email to supporters on Saturday morning, Shadow Brexit Secretary Keir Starmer said the letter "asks them to honour our shared democratic values, it asks them not to turn away from us now and deny us the chance for a final say".
"Add your name to the letter now and send a message to the powerful."
( With inputs from IANS )