Sevastopol city council approved nearly unanimously a resolution saying the port was legally Russian territory, citing a June plebescite in which residents voted to return the city to Russia.
The vote was certain to infuriate Ukrainian leaders who have lobbied against pro-Moscow sentiment in Crimea, former summer playground of Communist leaders where two-thirds of residents are ethnic Russians.
"Sevastopol was, is and will be a Russian city. Give us back our motherland. We want to go home," city council member Raisa Telyatnikova said during the debate.
Crimea and the Black Sea fleet make up one of the most tender spots in uneasy relations between Russia and Ukraine -- the two Slav giants forming the backbone of the Commonwealth of Independent States.
Separatism fuelled by Ukraine's economic decline prompted the election last January of Russian nationalist Yuri Meshkov as Crimea's first president. The local parliament passed a constitution seen by Kiev as the first step to secession.
The council also approved the text of a letter to Russian and Ukrainian leaders explaining their position and asking them to work out the city's status.
"The Sevastopol city council asks you to make a decision about the Russian status of Sevastopol and at last to resolve the problem of the Black Sea Fleet," the letter said.
"The city council's decision is the will of city residents and Black Sea Fleet sailors, 89 percent of whom voted in favour of Sevastopol as the base for the Russian Black Sea Fleet."
In Kiev, the chairman of Ukraine's parliament warned Crimea against any move to break up the country in a speech marking the third anniversary of independence from Moscow.
"We will respect the autonomy of regions, but will oppose the emergence of separatism in any form," Olexander Moroz told a glittering ceremony.
The Crimean peninsula was given to Ukraine as a "gift" in 1954, but many Russians see Sevastopol as Russian territory because of its military history.
In July 1993, the Russian parliament, dissolved after last October's armed revolt, declared Sevastopal a Russian city to the fury of Kiev and embarrassment of President Boris Yeltsin. Russia and Ukraine have been unable to resolve the issue of splitting the joint fleet.
The two sides are negotiating a treaty on friendship and cooperation and hope to sign the document when Yeltsin visits Ukraine on Nov. 29. Yeltsin has said the fleet issue must be resolved before he will sign such a treaty.
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