The demonstration comes as Prime Minister Mariano Rajoy warned that he won't rule out suspending Catalonia's regional autonomy to stop them from declaring independence.
"The ideal would be not to have to take drastic measures," he said in an interview published in daily El Pais.
People are set to travel from across Spain to the Catalan capital for the rally which will be held exactly one week since the contested vote that has triggered Spain's worst political crisis in a generation.
The Barcelona protest -- organised by the Societat Civil Catalana, the main anti-independence group in Catalonia -- gets underway at noon (10H00 GMT).
Organisers of the demonstration say they represent the "silent majority" of Catalans who oppose independence. Their slogan for the rally is: "Enough, lets recover good sense!"
Tens of thousands of demonstrators, many dressed in white, hit the streets of Madrid and other cities across Spain on Saturday to demand dialogue to end the dispute.
Tensions soared after police cracked down on voters during the banned October 1 Catalan independence referendum, prompting separatist leaders to warn they would unilaterally declare independence in days.
Tentative signs emerged Friday that the two sides may be seeking to defuse the crisis after Madrid offered a first apology to Catalans injured by police during the vote.
But uncertainty still haunts the country as Catalan leaders have not backed off from plans to declare the region independent.
Catalan leader Carles Puigdemont had been due to appear at the regional parliament on Monday but postponed it by a day, a spokesman said.
It remains unclear what he plans to say, although some separatist leaders hope he will use the opportunity to make a declaration of independence.
Spain could respond by suspending the region's existing autonomous status and imposing direct rule from Madrid.
"I don't rule out anything," Rajoy told El Pais in an interview published Sunday when asked about applying the constitutional provision that allows the suspension.
"But I must do things at the proper time... I would like the threat of an independence referendum to be withdrawn as quickly as possible.
Rajoy in the interview assured Catalan leaders that there "is still time" to backtrack and avoid triggering a tough response from the central government in Madrid.
'Break a people'
The crisis has raised fears of unrest in Catalonia, northeastern region about the size of Belgium that is home to 7.5 million people that accounts for a fifth of Spain's economy.
Angelo Rossini Calvo, 38, said he planned to attend the protest in Barcelona because he felt the separatist lawmakers did not have a big enough majority in the Catalan parliament to call the referendum.
"You can't call an important referendum like this, break a people and a country because you have one seat more," the cabin crew instructor told AFP at his flat in central Barcelona on the eve of the demonstration.
Pro-separatist lawmakers won a narrow majority of 72 seats in the 135-seat Catalan parliament in a regional election in September 2015 billed as a proxy vote on independence, capturing 47.8 percent of all ballots cast.
They then pushed through a law paving the way for the independence referendum held on October 1 with little debate in parliament despite rulings from Spain's Constitutional Court declaring such a vote illegal.
The Catalan government on Friday published final results from the referendum indicating that 90 percent of voters backed the idea of breaking away from Spain.
Turnout was 43 percent as Catalans who reject independence largely boycotted the polls.
The vote was not held according to regular electoral standards, without regular voter lists or observers.
Businesses and the government have kept up economic pressure on Catalonia, with several big companies announcing moves to shift their headquarters to other parts of Spain.
Rajoy's government passed a decree on Friday to make it quicker for businesses to shift their legal domiciles away from one region to another.
Recent polls had indicated that Catalans are split on independence, though leaders said the violence during the referendum turned many against the state authorities.
With its own language and cultural traditions, demands for independence in Catalonia date back centuries but have surged during recent years of economic crisis.