Ovarian cancer is one of the most prevalent malignancies in women. Screening of the disease is done using variety of biomarkers. Diagnostic performance of current biomarkers of the disease such as human epididymis protein (HE4) and CA125 shows contradiction in previous studies. The goal of this study was to evaluate serum levels of CA125 and HE4 in Iranian patients with ovarian cancer and compare specificity and sensitivity of HE4, CA125 and HE4 + CA125 in patients with different stages and diverse histology. To evaluate CA125 and HE4, 32 patients and 34 healthy women were selected. Origin of ovarian cancer was verified by expert gynecological oncologist. Significance and diagnostic performance were determined by ANOVA and receiver operator characteristic (ROC) and areas under the curve (AUC), respectively. Serum levels of CA125 and HE4 were significantly increased in patients in comparison with control group, especially for tumor cells originated from epithelium (p < 0.001). ROC-AUC for HE4, CA125 and HE4 + CA125 were 0.91, 0.86 and 0.91, respectively. Specificity of HE4 was more than CA125 (85 vs. 80 %). Conversely, sensitivity of CA125 was higher in comparison with HE4 (90 vs. 80 %). It is being noticed that cutoff point of HE4 and CA125 was 150 pmol/L and 38 U/mL, respectively. HE4 is slightly more specific for diagnosis of early stages of the disease, but the difference is not remarkable. CA125 and HE4 + CA125 have some diagnostic performance for prediction of advanced stages. Generally, the data of present study suggest that combining of HE4 and CA125 is a better screening tool for diagnosis of ovarian cancer.