Objective: Human Epididymis 4 (HE-4) protein, a new candidate for ovarian cancer detection shows promising diagnostic value for ovarian cancer diagnosis, this study aimed to assess the diagnostic significance of combined Human Epididymis 4 and Carbohydrate Antigen 125 (CA-125) serum protein levels in ovarian cancer detection. Subjects and Methods:A clinical case control study include; forty nine subjects; patients with ovarian cancer (n=33), non-cancer control group (n=16). Serum protein levels of HE-4 were measured using an enzyme linked immune sorbent assay (ELISA). All data were analyzed by SPSS software (version 21.0.0; IPM SPSS, Chicago, IL, USA). Results:The results showed that increased serum protein concentration of HE-4 (pMol/L) and CA-125 (U/ml) in the ovarian cancer group mean (SD)/median (range) 329.61±336.55/199 (28.72-1064) and 521.36±572.60/287 (10.50-2377), than non-cancer control group 64.80±38.51/54.53 (21 -160) and 28.35±10.80/28 (10-50) respectively (p<0.05). The receiver operating characteristic curve, the area under the curve (ROC-AUC) (95%CI) values of serum HE-4 was 0.85 (0.74-0.95). The sensitivity and specificity of HE-4 in diagnosing malignant ovarian tumors was 84.84% and 75.00%, respectively at ideal cutoff point that provided the best and highest accuracy in our own study and its level was related to clinical staging. Conclusion:Our study concludes that HE-4 protein a new candidate biomarker has a comparable diagnostic value for ovarian cancer detection compared to CA-125 with better performance of HE4 in our selected group of patients and combined serum protein levels of HE-4 with CA-125 improve the sensitivity obtained with only one tumor marker and their level was related to clinical staging. But, due to, "abnormally high concentrations of CA-125 found in many conditions and little is known about HE-4 diagnostic specificity till now in other benign and malignant conditions", both markers can be used for diagnosis and follow-up of patients taking in consideration their diagnostic specificity.