Analysis on the source of CO is very important for prevention of coal spontaneous combustion. In this article, a method of identifying primary CO in coal seams based on an oxygen isotope method is proposed through research. Tests on the oxygen isotopes (O-18) of CO were generated in coal oxidization reaction and coal pyrolysis reaction. It is found that O-18 of CO will reduce with rising temperature in both reactions, with O-18 of CO being 15-20 parts per thousand in air atmosphere and that being 25-28 parts per thousand in argon atmosphere in the temperature range of 130-220 degrees C, confirming that the sources of oxygen atoms of CO vary with different atmospheric conditions. Through collecting and testing a number of gas samples from the coal seam and roof in the coal mine, their O-18 of CO is found to be 35.2-37.3 parts per thousand. The result would improve the prevention of coal spontaneous combustion.