The students, many draped in red and yellow Catalan independence flags, gathered outside a building at the University of Barcelona in the centre of the Catalan capital.
"We will vote!" and "Independence!" they chanted as they marched along the Gran Via, one of Barcelona's main avenues, blocking traffic.
"The majority of young people are separatists, and if they weren't, they have become separatist after seeing what Spain has done in recent weeks," 16-year-old high school student Aina Gomez told AFP.
Over the past few days, judges and prosecutors have ordered the seizure of electoral material including millions of ballot papers, the closure of websites linked to the vote and the detention of key members of the team organising the referendum.
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The electoral board set up to oversee the vote has been dissolved, and on Tuesday prosecutors ordered police to seal off places to be used as polling stations and guard them until Sunday.
"If such a large number of people as exists in Catalonia want to separate from the country, they have to be allowed to vote," said 15-year-old student Pau Cabrinety.
Some students have said they may occupy schools and universities that could be used as polling station in Sunday's referendum in the wealthy northeastern region of Spain which is home to some 7.5 million people.
Catalonia's regional police force, the Mossos d'Esquadra, warned yesterday that there was a risk of a "disruption of public order" if police sealed polling stations as they have been instructed to do.