Chinese foreign ministry spokeswoman Hua Chunying said China conducted a "test flight" to check whether the airfield facilities met the standards for civil aviation.
Hua said the test flight was civilian in nature, adding that the "relevant activity falls completely within China's sovereignty".
"China has indisputable sovereignty over the Nansha Islands and their adjacent waters. China will not accept the unfounded accusation from the Vietnamese side," she said, referring to the Spratly Islands by their Chinese name.
The South China Sea is rich in natural resources. It is also a major shipping lane. Over half of the world's commercial shipping passes through the Indo-Pacific waterways - including one-third of the world's liquefied natural gas.
China claims almost the whole of the South China Sea, resulting in overlapping claims with several other Asian nations like Vietnam and the Philippines, Malaysia and Brunei.
They accuse China of illegally reclaiming land in contested areas to create artificial islands with facilities that could potentially be for military use.
In Hanoi, the foreign ministry said it has handed a protest note to China's embassy and asked Beijing not to repeat the action.
It described the flight "a serious infringement of the sovereignty of Vietnam on the Spratly archipelago".
Meanwhile, the United States has said it was concerned that Saturday's flight had exacerbated tensions.
Pooja Jhunjhunwala, a spokeswoman for the US State Department, said there was "a pressing need for claimants to publicly commit to a reciprocal halt to further land reclamation, construction of new facilities, and militarisation of disputed features".
Satellite images published by IHS Jane's Defence Weekly in April showed China building the airstrip on reclaimed land on Fiery Cross Reef in the Spratly Islands.
The landmass could accommodate a runway about 3,000m long, the report said.
It also showed dredging to the south of the reef, in apparent work to improve the reef's port facilities.
China says its work is legal and needed to safeguard its sovereignty.
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