Background: Breast cancer (BC) is the most common cancer in women and is the second most common cause of fatality in patients with cancer in the world. Tumor biomarkers have significant role in diagnosis and predicting the prognosis and decide the specific therapy to each patient. Aim: This study aims to evaluate the value of serum Ki-67 and Human epididymis protein 4 (HE4) as a diagnostic and prognostic markers in breast cancer patients and to analyze the associations these markers and their clinicopathological parameters. Subjects & Methods: This study included 120 patients with breast cancer and 30 healthy served as controls. We measured the serum level of Ki-67 and HE4 by ELISA technique. Results: Our results showed that there were statistically significant differences in serum Ki-67 and HE4 levels between two groups. Also, in breast cancer patients, there was significant difference between Ki-67 level, the same results with HE4 level and patient's age, tumor size, lymph node and metastasis. No significant relationship was found between Ki-67 level and tumor grade, ER, PR and HER2. The cutoff value for the prediction of breast cancer was determined at >2.48 ng/ml for Ki-67 and >25.2 pmol/L for HE4 with a sensitivity of 98.3%,100 %, specificity of 96.7%, 96.7%, positive predictive value of 98.3 %, 98.3%,  negative predictive value of 96.7 %, 100%  and accuracy of 88 %, 98.9% respectively. In conclusion: serum Ki-67 and HE4 can be used as strong diagnostic markers for breast cancer.