ABSTRACT: Background The present study was designed to verify the accuracy of the noninvasive biomarkers enolase/Cr, CA125, and CA19-9 as a clinical diagnostic tool for the detection of endometriosis. Methods A cross-sectional study was performed at Rasool-e-Akram Hospital affiliated to Iran University of Medical Sciences, Tehran, Iran, from April 2015 to April 2018. Eighty-six women were scheduled to undergo laparoscopy due to chronic pelvic pain, infertility, pelvic mass, and abnormal uterine bleeding. Serum and urine samples of all patients were collected preoperatively. Serum levels of CA125 and CA19-9, and urine levels of enolase-1 were measured. Serum levels of CA125 and CA19-9 were determined by the electrochemiluminescence method (ECL). Urinary enolase-1 was measured by the ELISA method. Result Serum levels of CA125 and CA19-9 were significantly higher in the endometriosis group than in controls (p < 0.001, p = 0.004, respectively). Levels of enolase I and enolase/Cr were higher in patients with endometriosis, but the differences were not statistically significant. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of combined enolase/Cr, CA125, and CA19-9 were 65%, 66.6%, 71%, and 60.1%, respectively. The positive likelihood ratio (PLR) and negative likelihood ratio (NLR) of combined enolase/Cr, CA125, and CA19-9 was 1.94 and 0.52, respectively. The area under the ROC curve for enolase/Cr + CA125 + CA19 ? 9 was 0.675 (95% confidence interval 0.573-0.710). Conclusion The present study revealed that concurrent measurement of enolase-1, CA125, and CA19-9 might be a valuable noninvasive test for the identification of endometriosis.