<p>Background: Prostate cancer is the second most common cancer in men. Prostate-Specific Antigen (PSA) is a tumor-associated glycoprotein with enzymatic activity which is secreted by the prostate gland. Following entry to the blood, 70-90% of PSA forms complexes with protease inhibitors and its enzymatic activity is inhibited. The serum level of PSA is increased and the rate of free PSA (fPSA) to total PSA is decreased in prostate cancer patients. Therefore, measurement of PSA and fPSA in serum is very valuable for diagnosis and prognosis of prostate cancer.<br /> Methods: In the present study, five anti PSA monoclonal Antibodies (mAb) were characterized by Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. For designing a sandwich ELISA, epitope specificity of these antibodies was studied by a competition ELISA. Free PSA was purified by electroelution technique from seminal plasma and used to produce polyclonal anti-fPSA antibody in rabbit. Purified polyclonal antibody (pAb) and mAbs were conjugated with HRP enzyme and Biotin (Bio) to set up the sandwich ELISA.<br /> Results: Three of the mAbs were found to recognize PSA similarly. One of these mAbs (2G3) was paired with anti-fPSA pAb to detect fPSA in serum. Eventually, serum fPSA concentration of 356 subjects was measured and compared by our designed ELISA and a commercial ELISA kit. Our results demonstrated a significant correlation (r=0.68; p&lt;0.001) between the two assays. Sensitivity and specificity of our designed ELISA was 72.4 and 82.8%, respectively.<br /> Conclusion: These results imply suitability of our designed ELISA for detection of fPSA in patients with prostate cancer.</p>