Taipei has repeatedly called for four new flight paths to be cancelled since China launched them earlier in January, but their complaints have fallen on deaf ears on the mainland.
Authorities on the island said they had not been consulted over the routes and described their introduction as "reckless", endangering flight safety, and politically motivated.
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China Eastern Airlines and Xiamen Air have since requested to operate 176 additional flights between Taiwan and China during the Lunar New Year period in mid-February.
But Taiwan's Civil Aeronautics Administration (CAA) said Friday it had refused permission because the two airlines had been using the controversial new routes, even though the extra Lunar New Year flights they requested would not have flown along them.
It is the first action taken against Chinese airlines over the row.
"The two airlines' requests are not approved for now because they are operating other flights using the M503 and related routes that are launched without a consensus between the two sides," a CAA official told AFP.
M503 is an existing route introduced in 2015, which also sparked a backlash from Taiwan, prompting Beijing to move it closer to the mainland and use it only for north-to-south flights.
The CAA official declined to comment on reports that around 50,000 passengers would be affected by the decision.
Beijing sees self-ruling democratic Taiwan as part of its territory to be brought back into the fold and has recently upped military drills around the island.
Cross-strait ties have steadily deteriorated since 2016 when Beijing-sceptic president Tsai Ing-wen took the reins. Chinese authorities unilaterally stopped communication between the two sides after she took office.
China said it had introduced the new flight routes to help ease congestion in its airspace over the strait.
The Civil Aviation Administration of China (CAAC) said the routes would only be used for civilian flights and China would maintain technical communications with Taiwan.
The CAAC added that the M503 route would also now be used for south-to-north flights, overriding the original restriction.
Taiwan's military has said it would intercept, warn and repel if necessary any planes that cross into Taiwanese airspace and threaten the island's security.
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