Chinese military personnel were put on "high alert" during the incident on 10 December, and issued warnings to leave.
The Pentagon said it was looking into the complaint.
China claims large swathes of the South China Sea but is in territorial dispute with a host of regional neighbours.
In October, China rebuked the US after a destroyer sailed close to a reef.
On Saturday, a statement from China's defence ministry accused the US of deliberately raising tensions in the area with the B-52 over-flight of the disputed Spratly Islands, which it calls Nansha.
A report in the Wall St Journal said there were two B-52s on the mission and that one unintentionally flew within two nautical miles of Cuarteron Reef on the Spratly Islands, possibly due to bad weather.
China said the flights "constitute a serious military provocation and are rendering more complex and even militarising conditions in the South China Sea".
It urged the US to take measures to prevent similar incidents.
The US has not taken sides on sovereignty issues in the area but has a "freedom of navigation" policy asserting right of passage for its military.
However, Pentagon spokesman Cmdr Bill Urban said the B-52 over-flight was not part of this policy, which analysts say might suggest a navigation error.
Regional tension
B-52s flew close to the Spratly Islands in November. The US said this mission was "fully in accordance with international law".The South China Sea region continues to cause tension in US-China relations.
The US guided missile destroyer USS Lassen sailed within 12 nautical miles of Subi Reef in late October, which the Chinese branded "extremely irresponsible."
Subi is one of the reefs China has built into artificial islands to help stake its claim to sovereignty.
However, the UN does not recognise such artificial islands as being part of the 12-nautical mile sea limit nations can claim for sovereign territory.
The recent decision by the US to approve a new arms package to Taiwan has also angered China.
CREDIT: BBC
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