"The Supreme Council deputies of Crimea have univocally fulfilled the decision of the Crimeans, they voted for holding the referendum on March 16, and no one is able to cancel it," Sergei Aksyonov said on Saturday.
On Thursday, Crimea's parliament voted to join Russia and set a referendum for March 16.
The conflict resulted from the overthrow last month of President Viktor Yanukovich after protests in Kiev that led to violence.
European Union leaders and the US President Barack Obama have said the referendum plan is illegitimate and would violate Ukraine's constitution.
But Aksyonov said the local government would go ahead with the public vote.
Aksyonov said the referendum was called at such short notice to "avoid provocations, as the situation in Ukraine is quite tense."
Meanwhile, authorities in Sevastopol have begun preparations for the referendum.
A working group has been set up and a 17-member election commission appointed.
The acting head of Sevastopol’s ruling council, Dmitry Belik, says international observers are welcome to witness and verify the process.
“If anybody wants to come and see how Crimean and Sevastopol residents vote, let them come and see. Nobody will be able to dispute the legitimacy of the election (referendum),” said Belik.
RA/MB