Abstract: To express, purify, and identify humanized anti-gamma-seminoprotein (γ-sm) Fab antibody screened from phage antibody library and to use it for antibody directed enzyme prodrug therapy for prostate cancer in the future. Methods: Recombinant phagemid DNA ( γ-sm-hFab/pComb3X ) was transformed into E. coli for soluble expression with IPTG induction followed by purification with immunoaffinity chromatography. The relative affinity and binding epitope of purified γ-sm-hFab were determined by inhibitory ELISA and competitive inhibitory ELISA respectively. The cellular and tissue binding specificity of purified γ-sm-hFab were validated by competitive flow cytometry and immunohistochemistry staining respectively, based on tissue microarray. Results: SDS-PAGE showed that the gene of humanized anti-γ-sm Fab antibody was successfully expressed in soluble form and was well purified. Smartview scanning indicated that the quantity of the expression product accounted for 20.5% of the concentrated bacterial total soluble protein. Western blot analysis was used to confirm further the purified product of this Fab antibody specifically bound to γ-sm, regardless of whether it was deglycosylated or not. ELISA inhibition curves demonstrated that the parent binding constant (Ka) is 0.78×108 M-1, which is about 37.5% of the relative affinity in the parent E4B7 mAb. Competitively inhibitory ELISA indicated that the humanized Fab antibody and parental E4B7 mAb can recognize the same antigen epitope. Flow cytometry showed significant binding of the humanized Fab antibody to LnCap and PC3M cell surface. This specific binding was further confirmed by immunocytochemical staining with the strongest immunoreactivity both on HGPIN and on Gleason 3 prostatic adenocarcinoma tissues. Conclusion: Succcessful prokaryotic expression, purification, and immunobiological binding analysis of the selected humanized anti-γ-sm Fab antibody lay the foundation for the optimal antibody-directed enzyme prodrug therapy for prostate cancer in the near future.