Abstract: A reliable metabolomics method for investigating intracellular metabolites in Lactobacillus paracasei by gas chromatography-mass spectrometry was developed.The effect of different solvents (pyridine, tetrahydrofuran and dimethylformamide) , and derived reagents (MTBDSTFA, BSTFA+TMCS and MSTFA) , for peak signal intensity and numbers were investigated.The best condition with pyridine as solvent and BSTFA+ TMCS as derived reagent was determined.Total 38 kinds of intracellular metabolites were identified in the samples of Lactobacillus paracasei.The method had been successfully employed in fermentation process study.Several biomarkers had been detected and identified.