The runway on Da Chu Thap (Fiery Cross Reef) is one of three China has been building for more than a year by dredging sand up onto reefs and atolls in Vietnam’s Truong Sa (Spratly) Islands.
Hanoi's claim to the area overlaps that of Beijing, which claims almost the whole of the East Vietnam Sea.
Two large Chinese civil aircraft landed on Wednesday on an airfield that China "illegally" built on the reef, Vietnam said.
"This is a serious violation of Vietnam's sovereignty, threatening peace and regional stability, threatening security, safety and freedom of navigation and aviation in the East Sea," foreign ministry spokesman Le Hai Binh said, using the name by which Vietnam refers to the contested sea.
"Vietnam resolutely requests China to immediately end similar acts and have no further violating acts," Binh said in an online statement.
Vietnam would defend its sovereign rights and jurisdiction in the area through peaceful measures, in line with international law and the United Nations charter, he added.
On Saturday, China landed a civilian plane on the same 3,000-metre (10,000-foot)-long runway in its first test in Truong Sa, which was also the first time it had used a runway in the area.
The United States, which has criticised China's construction of islands in the East Vietnam Sea, said after the first landing it was concerned that the flight had exacerbated tension.
Each year, more than $5 trillion of world trade is shipped through the East Vietnam Sea, where Brunei, Malaysia, the Philippines, Vietnam and Taiwan have rival territorial claims.