Objective To solve the problems on efficiency in chromatographic separation and life of chromatographic column in assay method of acetic acid, Methods After culture solution acidification, acetic acid was extracted by acetone and ether, and detected by gas chromatography. Results RSD varied from 1.7 % to 4.1%, recovery rate varied from 85.8 % to 94.6%. Conclusion The method was quick, simple and sensitive, and it can be used in strain classify and appreciation.