Purpose: To generate McAb against HBV M protein by gene immunization. Methods: Eukaryotic expression recombinant pcDNA3-PreS2/S containing HBV M protein coding sequence was constructed. Female BALB/c mice were immunized with the plasmid for preparation of specific McAb. Results: The 875 bp PreS2/S gene was correctly inserted into pcDNA3. M protein could be detected in the supernatant of transfeced cells, local injected sites of musle and spleen. One hybridoma cell line 2D3 that could consistently produce anti-M protein monoclonal antibodies were obtained. Conclusions: Monoclonal antibodies against HBV M protein could be produced by gene immunization.