In a separate order on Wednesday, the Constitutional Court told regional government officials they had 48 hours to show how they were preventing the vote from going ahead.
Many supporting the referendum argue that full sovereignty for Catalonia will benefit the region, especially amid the high unemployment rate and austerity measures that followed Spain's economic crisis, Jonathan Shafi, chairman of Radical Independence Campaign (RIC), told RT earlier this week.
The question for October 1 is what happens when the regional police (Mosos) is ordered by a court to stop the referendum, they refuse, and the Civil Guard is called in.
Over 700 municipal leaders pledged to help facilitate the vote, although the mayor of Barcelona has not yet taken a definitive position.
Prosecutors have already launched an official complaint against Catalan President Carles Puigdemont and members of his government over their referendum plans, accusing them of civil disobedience, misfeasance and misappropriation of public funds - the latter carrying jail sentences of up to eight years.
Million march for Catalonian independence
Prime Minister Mariano Rajoy, whose conservative government is fiercely against the vote, wished Catalonia "a good day", calling "for a Diada of freedom, cohabitation and respect for all Catalans".
"What is happening is that people are looking to have more control over their lives; they are looking for much more in terms of a say in how their lives are run", he said.
"If anyone urges you to go to a polling station, don't go, because the referendum can't take place, it would be an absolutely illegal act", Mr Rajoy said. It argues Spain's 1978 constitution stipulates that regional governments can not call an independence referendum.
The economically powerful Catalonia has a thriving population of 7.5 million and accounts for a fifth of Spain's economic output.
Hundreds of thousands of Catalans rallied on Monday in Barcelona on their national day to demand their region's secession from Spain and support the right to vote.
Spain's economic troubles and a perception that the region pays more in taxes than it receives in investments from Madrid have helped push the secessionist movement.