Prostate-specific antigen (PSA) is a glycoprotein that is secreted by prostate gland epithelial cells, and elevation of PSA level in serum is often observed with prostate cancer or prostate disorders. Therefore, detection of PSA level in patient specimens is used as a diagnostic marker when screening of prostate cancer. In this study, we developed PSA antibody-conjugated microsphere beads that can be used for quantitation of PSA. We optimized the procedure for bead preparation and validated the detection analysis by using LNCaP and DU-145 prostate cancer cell lysates. By applying the procedure, extracellular PSA from culture media of LNCaP cells and standard PSA proteins were quantified to assess whether the antibody-conjugated microsphere bead can be used to detect trace amounts of PSA. The PSA level results obtained by using the antibody-conjugated microsphere beads indicate that the procedure is sensitive and quantitative in analyzing PSA. Taken together, the results suggest that the method is suitable for microquantitation of PSA from patient specimens.