Madrid has pledged to stop the vote, declared illegal by the courts.
Dozens of national police vehicles have left their base in the port of the regional capital, Barcelona, as officers deploy to enforce the ruling.
Overnight, thousands of separatist supporters occupied the schools that were to be used as voting centres. Police say they will be evicted.
Referendum organisers had urged voters to turn up "to defend" the polling stations at 05:00 (03:00 GMT) and to wait for voting to start at 09:00. They called for peaceful resistance to any police action.
In some areas, farmers positioned tractors on roads and in front of polling station doors, Spanish media reported, and school gates were taken away to make it harder for the authorities to seal buildings off.
"I have got up early because my country needs me," said Eulalia Espinal I Tarro, 65, who was outside a Barcelona school.
"We don't know what is going to happen but we have to be here."
Sunday would be an "important date for democracy", regional Vice-President Oriol Junqueras told TV3, the main Catalan public channel.
On the eve of the vote, thousands of demonstrators calling for Spanish unity held rallies in cities across Spain, including in the Catalan capital Barcelona.
They waved Spanish flags and carried banners reading "Catalonia is Spain".
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