Bank of Spain governor Luis Linde said Catalonia would drop out of the eurozone if it broke away from Spain and would lose access to credit from the European Central Bank.
He followed European leaders, businesses and big commercial banks in warning of the financial risks of secession for the rich but heavily indebted northeastern region.
Nationalist leaders in Catalonia have vowed to declare independence within 18 months if they win the vote on September 27.
"Leaving the European Union means automatically leaving the eurozone," so banks in Catalonia would "stop having access to the ECB's facilities", he told reporters.
He said there was a risk, albeit a "highly unlikely" one, that Catalonia would end up having to impose banking restrictions as Greece did in June at the height of its debt crisis.
Catalan president Artur Mas, who is spearheading the independence push, criticised the Bank of Spain governor, saying it was "irresponsible and indecent to threaten things that nobody in a democratic country would dare insinuate. They do it because they have no other argument".
Spain's conservative central government, which fiercely opposes Catalonia's moves to break away to form its own country, denied accusations that the central bank was trying to scare voters with its warning.
"If my friends are going to jump off a bridge I will ask them not to jump. And if they jump it is no use to be waiting below to help them," Foreign Minister Jose Manuel Garcia-Margallo told reporters.
"Warning what might happen does not seem to me to be trying to frighten people, it is simply telling them to measure the consequences before they vote," he added.
The latest opinion polls show separatist parties could win a majority in the Catalan regional parliament on September 27.
The independence drive has intensified over the recent years of economic crisis. Catalans complain about how much of their tax money is redistributed to the rest of Spain.
Their vows to secede have posed a tough political challenge to the conservative central Spanish government. It is looking to strengthen Spain's economic recovery and preparing to fight a general election in December.
Catalonia, a region of 7.5 million people which accounts for a fifth of Spain's economic output, has its own language which was suppressed during General Francisco Franco 1939-75 dictatorship.
With an economy about the size of Portugal's, Catalonia houses global firms including toll road, telecoms and airport operator Abertis and healthcare group Grifols.