With tensions mounting, separatist organisations called for more people to protest as leaders in a region deeply divided over independence pressed ahead with preparations for the October 1st vote.
WATCH:Video footage from spontaneous protest on the streets of Barcelona.
Spain's conservative Prime Minister Mariano Rajoy called for calm, urging "a return to normality and a return to common sense."
But Catalan President Carles Puigdemont accused Madrid of imposing a "de facto" state of emergency to stop the referendum.
The opposition Socialist party's Catalan leader, meanwhile, asked the regional and central governments to "stop an escalation that is leading to a disaster."
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Among those arrested by the Guardia Civil police was Josep Maria Jove, secretary general of economic affairs and Catalonia's deputy vice president, a regional government spokesman said.
The others work in various Catalan government departments, including its economic and budget affairs departments, a local Guardia Civil spokesman said.
In addition, police on Wednesday seized "close to 10 million ballot papers" destined for the vote, the interior ministry said.
'They declared war'
In central Barcelona, police said close to 4,000 protesters gathered near Jove's office, many draped in red and yellow Catalan flags, chanting "Independence!" and "We will vote!"
Anna Sola, an unemployed 45-year-old, said she rushed out to protest after hearing about Jove's arrest on the news and through text messages from friends.
"They are attacking our institutions, those that we voted for, just for simply doing what the people want," she said.
An influential pro-independence citizens' organisation, the Catalan National Assembly (ANC), urged Catalans to hit the streets in protest.
"Let's go out to defend our institutions in a non-violent way. They made a big mistake, we wanted to vote and they declared war," its president Jordi Sanchez said in a statement.
Polls show that while Catalans are sharply divided on whether they want independence or not, a large majority would like to vote to settle the matter.
Threats and seizures
The police operation comes a day after officers seized a trove of documents related to the independence referendum from the offices of Unipost, a private delivery company, in Terrassa near Barcelona.
Police said they confiscated over 45,000 notifications which were about to be sent to Catalans selected to staff polling stations for the vote, representing 80 percent of the numbers necessary to ensure the stations were adequately staffed.
Madrid has also threatened to arrest mayors who facilitate a vote deemed illegal by the Constitutional Court, has seized posters and fliers that promote the plebiscite and has tightened control over the region's finances.
The constitution states that the unity of the Spanish nation is "unbreakable" and that only the central government has the power to call a referendum on any matter.
Separatists in Catalonia, a region with its own language and customs, have retorted they have a democratic right to decide on their future.
'Leading to disaster'
Tensions also reached the parliament in Madrid on Wednesday, where Gabriel Rufian, a lawmaker for the pro-separatist Catalan Republican Left (ERC), told Rajoy to take his "dirty hands off Catalan institutions".
"The will of the Catalan people cannot be stopped. And now we will leave to support our friends," he added before storming out of the assembly with fellow ERC lawmakers.
But Rajoy defended his government's position, saying it was "fulfilling its obligation".
In an interview broadcast Tuesday with Bloomberg television in New York, Spain's Foreign Minister Alfonso Dastis accused Catalan separatists of using "Nazi" tactics to intimidate their opponents before the referendum.
Pro-separatist parties captured 47.6 percent of the vote in a September 2015 Catalan election which was billed as a proxy vote on independence, giving them a narrow majority of 72 seats in the 135-seat Catalan parliament.
But a survey commissioned by the regional government in July showed that 49.4 percent of Catalans were against independence while 41.1 percent were in favour.