Carles Puigdemont risks losing his leadership as the vote, set for Sunday, has been declared illegal by the Spanish courts.
Enric Millo, the Spanish government’s representative in Barcelona, said he would “keep hoping until the last minute that the Catalan government has a change of heart and calls off the referendum”.
Spanish authorities have threatened referendum organisers with arrest and have ordered police to patrol polling stations in a bid to stop the vote.
Mr Millo said: “If that does not happen it is very likely that our interlocutors in Catalonia afterward will be different people.
“Today we can affirm that there will be no effective referendum in Catalonia. All the referendum’s logistics have been dismantled.”
Mr Millo’s comments mark a further escalation in the row over the two parts of Spain as Madrid attempts to stop the poll from going ahead.
Last week Spain‘s state police raided Catalan government offices, arrested senior officials and seized ballot boxes, papers and other electoral material.
Spanish prosecutors also threatened independence campaigners with sedition charges.
Despite this, the Catalan government has promised to go ahead with the independence referendum it says is binding.
Mr Puigdemont last month said he is willing to go to jail if it meant giving the Catalan people a vote.
He told the Financial Times: “I don’t want to go to prison but there is nothing they can do to me that will make me stop this referendum.”
The Catalan government has promised a declaration of independence within 48 hours if the majority of people vote yes on Sunday.
A declaration would mean Spain suffers its most serious constitutional crisis in more than 30 years.
Spanish Prime Minister Mariano Rajoy has staked his reputation on the vote not happening, despite facing serious criticism for allowing an informal ballot on independence in 2014.
The Spanish government earlier today said regional police will take control of any voting booths before the referendum.
Catalonia’s prosecutor has ordered the regional police – known as the Mossos d’Esquadra – to take control of any voting booths from Friday, a spokesman for the Madrid government’s Catalan delegation said.
In an order to police issued on Monday, the prosecutor’s office said they would take the names of anyone participating in the vote and confiscate relevant documents.
Anyone in possession of the keys or entrance codes to a polling booth could be considered a collaborator to crimes of disobedience, malfeasance and misappropriation of funds, the order said.
Senior Madrid government officials said on Monday authorities had done enough to prevent a meaningful referendum in the region as Catalonia lacked an election commission, ballots boxes, ballot papers, a transparent census and election material.
The government has in recent weeks taken political and legal measures to prevent the referendum by exerting more control over the use of public funds in Catalonia, arresting regional officials and seizing election leaflets.
Hundreds of police reinforcements, including the national Civil Guard, have been deployed in Barcelona and other cities.
Madrid has also threatened fines against bureaucrats working on the ballot, including the region’s election commission, which was dissolved last week.
These actions have provoked mass demonstrations and drawn accusations from Catalan leaders that the Madrid government was resorting to the repression of the Franco dictatorship.