Objective:To clone,express human Golgi protein 73 (GP73),develop and characterize the monoclonal antibody(mAb) against GP73,a valuable serum marker for hepatocellular carcinoma(HCC). Methods: GP73 gene was amplified by PCR,and cloned into prokaryotic expression vector pComb3XSS. The recombinant construct was expressed in Top10F′ E. coli. host,and purified with their fusion partner by HisFF Trap affinity chromatography. The recombinant protein was used to immunize the BALB/c mice for mAb development. The titer of the mAb was detected by ELISA and its specificity was analyzed by Western blot. The sera level of GP73 in HCC patients and healthy people was measured by co-immunoprecipitation(IP). Results: The GP73-6×His recombinant protein was successfully expressed and purified. Five hybridoma cell lines against GP73 were obtained. IP revealed that the mAb could combine with GP73 in human sera with high specificity. The level of GP73 in HCC patients is much higher than that of healthy people. Conclusion: The success in mouse a-GP73 mAb development provides the basis for further developing a sandwich ELISA for detection of human GP73.