Abstract: Objective To investigate the expression levels of serum miR-199b-5p and ring finger protein 213 (RNF213) in patients with polycystic ovary syndrome (PCOS) and to explore their clinical significance.Methods A total of 200 PCOS patients who were admitted to the Affiliated Hospital of Xuzhou Medical University from August 2024 to June 2025 were enrolled as a study group. Meanwhile, 100 healthy women undergoing physical examination were selected as a control group. According to their homeostasis model assessment of insulin resistance (HOMA-IR), body mass index (BMI), and serum total testosterone (T), the study group was further divided into an insulin resistance group (HOMA-IR ≥2.69, BMI <28 kg/m², and T <0.7 μg/L; n=33), an obesity group (BMI≥28 kg/m², HOMA-IR <2.69, and T <0.7 μg/L;n=35), and a hyperandrogenism group (T≥0.7 μg/L, HOMA-IR<2.69, and BMI<28 kg/m²;n=33). The remaining 99 PCOS patients with HOMA-IR < 2.69, BMI < 28 kg/m², and T<0.7 μg/L were regarded as a reference population, from which 33 cases were randomly selected each time as the reference group for the corresponding positive subgroup. Serum miR-199b-5p expression levels were measured by RT-qPCR, and RNF213 levels were measured by enzyme-linked immunosorbent assay (ELISA). The regulatory relationship between miR-199b-5p and RNF213 was verified by a dual-luciferase reporter gene assay. Pearson correlation analysis was used to evaluate the correlations of miR-199b-5p and RNF213 with clinical indicators, and receiver operating characteristic (ROC) curve were plotted to assess their diagnostic value.Results The study group showed significantly lower serum miR-199b-5p levels but higher RNF213 levels than the control group (P<0.001). Among the different subgroups, miR-199b-5p levels in the insulin resistance group, obesity group, and hyperandrogenism group were all lower than those in the corresponding reference groups, while RNF213 levels were higher than those in the non-characteristic groups (P<0.05). Dual-luciferase reporter gene assay confirmed that miR-199b-5p directly targeted and regulated RNF213. Furthermore, RNF213 was positively correlated with anti-Müllerian hormone (AMH), luteinizing hormone (LH), HOMA-IR, triglycerides (TG), and T (P<0.05), whereas miR-199b-5p was negatively correlated with these indicators (P<0.05). ROC curve analysis indicated that the area under the curve (AUC) for the combined detection of miR-199b-5p and RNF213 in diagnosing PCOS was 0.97, which was significantly higher than that of either marker alone (P<0.001).Conclusions Serum miR-199b-5p expression is downregulated and RNF213 expression is upregulated in patients with PCOS. Both are closely associated with glucose and lipid metabolism as well as hormone levels, and a targeting regulatory relationship exists between them. Combined detection of miR-199b-5p and RNF213 has high diagnostic value for PCOS.