Preterm labor is a serious concern that can lead to preterm birth, posing substantial risks for both the mother and the neonate. Despite approximately 15 million preterm births worldwide each year, there is a lack of sufficient strategies for predicting and preventing preterm labor. Here, we present a non-invasive method for simultaneously detecting exosomal miRNA and protein biomarkers in vaginal discharge, enabling early diagnosis of life-threatening conditions in both the mother and the neonate. Our non-invasive vaginal discharge biopsy using a swab enables the isolation of enriched intact extracellular vesicles through our microfluidic platform called Biologically-intact Exosome Separation Technology (BEST). We observed differential expression of specific miRNAs, including up-regulated hsa-miR-206 and down-regulated hsa-miR-3674, hsa-miR-365a-5p, and hsa-miR-193b-3p, in mothers experiencing preterm labor. We also found significant differences in protein expression in mothers with preterm labor compared to full-term mothers, indicating the involvement of HGS, ATL3, APOH, and GUSB in preterm labor mechanisms. We envision a future in which non-invasive detection of unique miRNA and protein biomarkers in vaginal discharge transforms global healthcare by enabling early detection and effective treatment of preterm labor.