The prediction and treatment of spontaneous preterm labor (sPTL) are critical challenges in clinical practice due to obscure etiology and are lack of highly specific and sensitive testing methods. In the current study, we extended the application of non-invasive prenatal testing (NIPT) into the field of sPTL prediction, and using high-throughput small RNA-sequencing technique to screen the potential biomarkers for sPTL in maternal peripheral blood. We found that hsa-miR-150-5p and hsa-miR-512-3p were specifically decreased in the sPTL patients, compared to either term labor or term not labor patients. The change of hsa-miR-150-5p is validated using quantitative PCR with the AUROC of hsa-miR-150-5p around 0.8272, suggesting it’s a promising biomarker for sPTL prediction. In the discovery set, hsa-miR-150-5p exhibited an AUROC of approximately 0.8508, which was validated in an independent cohort, accurately classifying preterm samples with an AUROC of 0.8010. Moreover, we showed that miR-150-5p inhibited migration and invasion of chorionic cells by directly targeting ADAM19, a member of ADAM (a disintegrin and metalloprotease domain) family. The significant increase of ADAM19 in chorionic tissues from sPTL patients further indicates its inverse correlationship with miR-150-5p. ADAM19 is a sheddase of membrane-bound TNF-a and releases the TNF-a trimer into the extracellular environment, which reciprocally induces the expression of ADAM19 to form a regenerative cycle and augments the migration and invasion of fetal membrane cells. Our results suggested that miR-150-5p is not only an effective non-invasive biomarker for sPTL, but also plays an important biological role in the premature rupture of fetal membranes associated with spontaneous preterm labor.