Abstract： Objective To establish and identify a new monoclonal antibody (MAb) against human hepatoceilular carcinoma (HCC) with high specificity and to provide evidence for targeted diagnosis and treatment of liver cancers. Methods Human HCC cell line HepG-2 was used to immunize BALB/c mice using the tail vein injection combined with intrasplenic injection. The sensitized splenic cells were fused with isogenic rat myeloma cells SP2/0-Ag14. The supernatant of the fused cells was detected by ABC immunohistochemistry (IHC) for primary antibody screening. The positive clones were subcloned using the limiting dilution method, and the chromosome of hybridoma was analyzed. The specificity of obtained MAb against HCC was identified by IHC. The epitope of the antigen was detected with laser confocal scanning microscope (LSCM). The subclass of the MAb was analyzed by enzyme linked immunosorbent assay (ELISA). The biodistribution of the MAb was detected in HCC-bearing nude mice to identify its biological characteristics. Results (1) One strain of hybridoma was obtained which produced antihuman HCC MAb with up to 98.5% (67/68) specificity in binding with liver cancer tissues. (2) After injection of 131I-MAb, radioactivity appeared increasingly stronger at the site of tumor and gradually weaker in the blood, liver, kidney and lungs. At 72 h, the ratio of radioactivity in tumor over blood and in tumor over liver was 15.76±3.28 and 7.23±1.70, respectively. Conclusions A novel MAb targeting well at HCC with high specificity is obtained. The MAb may be of potential use in diagnosis and treatment of primary HCC.