Abstract: Objective To investigate the expression of cysteine-rich protein 61 (CYR61) and microRNA-22-3p (miR-22-3p) in ovarian endometriosis (EMs) and their clinical significance.Methods A total of 40 ovarian EMs patients, who underwent laparoscopic surgery and pathologically diagnosed at the Affiliated Hospital of Xuzhou Medical University from January to August 2025, were selected. Ectopic cyst wall tissue (ectopic group) and normal endometrial tissue (in-situ group) were collected. Furthermore, 40 patients undergoing hysterectomy for uterine leiomyoma were selected as the control group, with normal endometrial tissue collected. Preoperative venous blood was collected from 40 EMs patients (study group) and 40 patients with uterine myoma (control group). CYR61 protein expression in tissues was detected by immunohistochemistry. MiR-22-3p and CYR61 mRNA expression in tissue and blood samples were examined by quantitative real-time PCR (qRT-PCR). The levels of CYR61 protein in blood samples were measured by enzyme-linked immunosorbent assay (ELISA). The correlations between CYR61, miR-22-3p, and CA125 with clinical and pathological features were analyzed. Receiver operating characteristic (ROC) curves were plotted to assess the diagnostic value of each indicator.Results In tissue samples, the positive expression rate and the levels of CYR61 mRNA and miR-22-3p in the ectopic group were significantly higher than those in the in-situ and control groups (P<0.001), with no significant difference between the in-situ group and the control group (P>0.05). The positive rate of CYR61 protein expression in the ectopic endometrial tissue was significantly higher than that in the in-situ and control groups (P<0.05), with no significant difference between the in-situ and control groups (P>0.05). In blood samples, the levels of plasma miR-22-3p and serum CYR61 levels in the study group were significantly higher than those in the control group (P<0.001). In ovarian EMs patients, CYR61 expression was positively correlated with miR-22-3p expression (r=0.543, P<0.001). ROC curve analysis revealed that the areas under the curve (AUC) for CYR61, miR-22-3p, CA125, and the combined diagnosis of the three were 0.830, 0.895, 0.919, and 0.990, respectively (all AUC >0.80). The diagnostic efficacy of the combined indicators was superior to that of any single indicator alone.Conclusions In EMs patients, the expression of CYR61 and miR-22-3p in blood and ectopic cyst wall tissue is significantly elevated, and their levels were positively correlated. These findings suggest that they may be involved in the pathological process of EMs. The combined detection of CYR61, miR-22-3p, and CA125 may provide new potential biomarkers for the clinical diagnosis and monitoring of endometriosis and offer new therapeutic targets for its treatment.