Oleksandr Turchynov told parliament it was being conducted “stage by stage, in a responsible… manner”.
Hours later, gunfire was heard at an airbase which officials said had been in the hands of militants.
Oleksandr Turchynov said the airbase at Kramatorsk had been “liberated” from “terrorists”.
Pro-Russian rebels have seized buildings in about 10 towns and cities across Ukraine’s eastern provinces, which form the heartland of Ukraine’s heavy industry.
Thousands of Russian troops are reported to be deployed along the border, kindling fears that any crackdown on the rebels could trigger an invasion.
Russia annexed the Ukrainian province of Crimea last month, after it broke away and held a controversial referendum on self-determination.
A crowd of some 200 people remained on Tuesday evening, chanting slogans in favor of a referendum on the region’s future.
A spokesman for the Russian foreign ministry expressed “deep concern” at reports of casualties in eastern Ukraine, but these could not be confirmed.
Oleksandr Turchynov said the aim of the operation in the east was to “protect Ukrainian citizens, to stop the terror, to stop the crime, to stop the attempts to tear our country apart”.
Protesters gathered outside parliament in Kiev to demand action against the separatists.
There were reports overnight of gun attacks on rebel checkpoints near the Donetsk town of Sloviansk, where pro-Russian militants seized a police station and a security services building at the weekend.
A police building in Kramatorsk was also seized but the militants there have reportedly now handed back control to the police.
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