In recent years, microbial metabolomics has gradually become a new analysis method. Although microorganisms have the characteristics of simple structure and single nutritional requirement, they also have many kinds of their own, and the physical and chemical properties of intracellular metabolites are different. Therefore, there are still a lot of deficiencies in the pre-sample treatment. Based on the analytical method of GC-MS, a metabolomics analysis method for yeast S288C was established in this paper. The effects of five solvents, including methanol (-20℃), acetonitrile/water (1:1) (-20℃), acetonitrile/methanol/water (2:2:1) (-20℃), water/isopropanol/methanol (2:2:5) (-20℃) and 75% ethanol (boiling) on the extraction of intracellular substances were investigated. The optimum solvent was water/isopropanol/methanol (2:2:5) (-20℃). The method could detect 348 metabolites, 142 metabolites are identified by NIST library searching, including 46 amino acids, 9 carbohydrates, 30 organic acids, 17 alcohols and 23 esters.