Background: To evaluate the effect of carbohydrate antigen 125 (CA125) and CA19-9 in distinguishing stage Ⅲ and Ⅳ endometriosis from benign and malignant tumors, and to explore whether it is related to the clinical features of the disease. Methods: In a retrospective cohort study based on clinical data from hospitals, a total of 183 patients with pathologically confirmed diagnosis of ovarian endometriotic cysts (OEC) in Hainan Provincial People’s Hospital for surgical treatment from January 2019 to August 2022 were selected as the case group, and a total of 276 cases of benign diseases, including 184 cases of benign ovarian tumors, 94 cases of gynecological common diseases, and 102 cases of malignant ovarian tumors were selected as the control group, with a total of 276 cases of benign diseases, including 184 cases of benign ovarian tumors, 94 cases of gynecological common diseases, and 102 cases of malignant ovarian tumors. There were also 23 cases of ruptured ectopic cysts. We compared the clinical characteristics (age of onset, fertility, dysmenorrhea, preoperative CA125 and CA19-9 values) of the patients in the OEC group with those of the other control groups; analyzed the serum CA125 and CA19-9 values in relation to the pathological characteristics of OEC (recurrence, unilateral and bilaterality, multilocularity and unilocularity, rupture, dysmenorrhea, fertility, and staging); and analyzed the CA125 and CA19-9 values by unordered logistic regression, CA19-9 to predict OEC; sensitivity, specificity and cut-off values of CA125, CA19-9 and their combined indexes to diagnose OEC. Results: The symptoms of dysmenorrhea and infertility in OEC group were significantly higher than those in the other three groups. The preoperative CA125 value in OEC group was higher than that in benign tumor and other gynecological diseases group, and significantly lower than that in malignant tumor group. There was no significant difference in the value of CA19-9 and CA125 in the degree of dysmenorrhea, recurrence and infertility. The values of CA19-9 and CA125 of multilocular cysts were higher than those of unicameral cysts, bilateral cysts were higher than unilateral cysts, and ruptured cysts were significantly higher than unruptured cysts. The value of CA125 in the dysmenorrhea group was higher than that in the non-dysmenorrhea group, and that in the fourth stage was higher than that in the third stage, and the difference was statistically significant (p 0.05). Unordered multicategorical logistic regression analysis determined that CA125, could be a predictor in the comparison of OEC with benign disease; in the benign control group the cut-off value for CA125 was >23.1 IU/mL with an area under the curve (AUC) value of 0.90 (0.869–0.926), a sensitivity of 89.62% and a specificity of 81.52%. In the malignant control group the cut-off value for CA125 was \le209.2 with an AUC value of 0.859 (0.813–0.897), sensitivity 95.08% and specificity 71.57%. Conclusions: The effect of serum CA19-9 in the diagnosis of Endometriosis (EMT) is not ideal. CA125 has a certain value in the diagnosis of endometriosis, but it is necessary to explore the range of cut-off value.