Preterm birth (PTB) is a social problem that adversely affects not only the survival rate of the fetus, but also the premature babies and families, so there is an urgent need to find accurate biomarkers. We noted that, among the causes, the change from eubiosis to dysbiosis of the vaginal microbiome leads to changes in metabolite composition. Therefore, in this study, cervicovaginal fluids (CVF) from 30 preterm birth and 60 term birth mothers were collected, and short-chain fatty acids (SCFA), organic acids, amino acids and other polar metabolite compounds were analyzed. Targeted analysis was performed by first extracting CVF with an organic solvent, derivatizing SCFAs with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide, and then analyzing them by gas chromatography-mass spectrometry (GC-MS). In residual aqueous CVF, the polar metabolites produced in the biochemical process were derivatized using methoxyamine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and analyzed by GC-MS to performed non-targeted analysis. As a result, 9 SCFAs including hextanoate were quantified, and 58 polar metabolites were detected in 90 clinical samples. The criteria of statistical analysis and detection rate of clinical sample for development of PTB biomarkers were presented, and 19 biomarkers were selected based on it, consisting of 1 SCFA, 2 organic acids, 4 amine compounds, and 12 amino acids. In addition, the model was evaluated as a suitable indicator for predicting PTB without distinction between sample collection time. We hope that the developed biomarkers based on microbiota-derived metabolites could provide useful diagnostic biomarkers for actual patients and pre-pregnancy. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number 2021R1A6A1A03044296)