The strike follows Sunday's violence that erupted when Spanish police beat people trying to vote in Catalonia's independence referendum.
Large numbers of Catalans are expected to observe a general strike on Tuesday to condemn police violence at a banned weekend referendum on independence, as Madrid comes under growing pressure to resolve Spain's worst political crisis in decades.
Violent scenes played out in towns and cities across Catalonia region on Sunday as riot police moved in on polling stations to stop people from casting their ballots, in some cases charging with batons and firing rubber bullets to disperse crowds.
Flights and train services could be disrupted on Tuesday as well as port operations after unions called for the stoppage to "vigorously condemn" the police response to the poll, in which Catalonia's leader said 90 percent of voters backed independence from Spain.
Barcelona's public universities are expected to join the strike, as is the contemporary art museum, football club FC Barcelona and the Sagrada Familia, the basilica designed by Antoni Gaudi and one of the city's most popular tourist sites.
"I am convinced that this strike will be widely followed," Catalan leader Carles Puigdemont said ahead of the protest.
UN rights chief Zeid Raad al Hussein said he was "very disturbed" by the unrest while EU President Donald Tusk urged Madrid to avoid "further use of violence."
The European Parliament will hold a special debate on Wednesday on the issue.
"We call on all relevant players to now move very swiftly from confrontation to dialogue. Violence can never be an instrument in politics," European Commission spokesman Margaritis Schinas said, breaking weeks of virtual EU silence on the Catalan issue.
Residents in many cities briefly stopped work at midday on Monday and descended onto the streets in silent, solemn protest.
In Barcelona, municipal police said about 15,000 people stopped traffic as they rallied, many draped in the blue, yellow and red Estelada flag used by Catalan separatists, shouting "the streets will always be ours."
"This was the norm under Franco!" the crowd chanted, referring to former dictator Francisco Franco whose 1939-75 regime repressed Catalan language and culture.
The government of Prime Minister Mariano Rajoy held emergency talks after Puigdemont declared Sunday that Catalonia had "won the right to an independent state."
Puigdemont has appealed for international mediation to help solve the crisis and called for police deployed to Catalonia from other parts of Spain for the vote to be removed.
The regional government said 2.26 million people took part in the poll, or just over 42 percent of the electorate.
But any attempt to unilaterally declare independence is likely to be opposed, not just by Madrid, but also a large section of the Catalan population, a region of 7.5 million people that is deeply split on the issue.
Puigdemont has said he will now present the results to the region's parliament, where separatist lawmakers hold a majority, and which has the power to adopt a motion of independence.
The Catalan leader said close to 900 people had received medical attention, though regional authorities confirmed a total of 92 injured. Four were hospitalised, two in serious condition.