Intestinal microbiota and its main metabolites， short-chain fatty acids (SCFAs)， are considered to be important factors for intestinal environmental stability and metabolic diseases. Gas chromatography-mass spectrometry (GC-MS) is the most commonly used method for the analysis of short-chain fatty acids in biological samples. Trimethylsiliconyl (TMS) derivatization reagents N，O-bis (trimethylsiliconyl) -trifluoroacetamide (BSTFA) are commonly used to improve the sensitivity and accuracy of gas chromatography/mass spectrometry. We used BSTFA to analyze SCFAs in feces， cecum and colon contents by GC-MS. Selective ion monitoring mode was used for quantitative analysis of short-chain fatty acids with high sensitivity. At 37 °C， SCFAs derived from BSTFA showed good linearity (R2>0.998)， recovery 86%-96% and precision RSD<4%. The results showed that GC-MS combined with BSTFA derivatization can be successfully applied to quantitative analysis of SCFAs in feces， and it is an accurate and universal method.