The statement followed reports that Tsai will transit through Hawaii and the US territory of Guam next week -- a trip that comes at a delicate time with President Donald Trump slated to visit Beijing in November.
"As for the Taiwan leader stopover in US territory, her true motives for transiting by the US is self-evident," foreign ministry spokesman Geng Shuang said during a regular press briefing.
"We hope that the United States would abide strictly by the 'one China' policy... deny her permission to stopover, avoid sending the wrong signals to Taiwan independence forces and take concrete actions to preserve the overall state of US-China relations."
Beijing has been wooing other countries to dump Taiwan, which it sees as part of its territory to be brought back into its fold at some point.
The two sides split after a civil war in 1949, and while Taiwan sees itself as a sovereign nation, it has never formally declared independence.
The island has been a thorny issue in China-US relations, as Trump began his transition into office by taking a precedent-breaking phone call from Tsai.
Tsai's last visit was to Central American allies in January, during which she made stopovers in the US.
Beijing had asked Washington then to bar Tsai from flying through US airspace, but the request was ignored.
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