Objective To prepare the monoclonal antibodies specific to subtype of H5N1 avian influenza virus and offer tool for correlative researches.Methods Monoclonal antibodies specific to subtype of H5N1 avian influenza virus were developed by fusing SP2/0 myeloma cell with spleen cell of BALB/c mice immunized with AIV_H5N1, the characterization of three McAbs can specifically react with AIV_H5N1 by hemagglutination inhibit test(HI), enzyme linked immunosorbent assay(ELISA) and immunofluorescence. The inmmunoglobulin subclass of the McAbs was then identified. Results Three colone hybridoma secreting McAbs against AIV_H5N1 were obstained as F8,F9,G11 respectively, the inmmunoglobulin subclass of the McAbs was identified as IgG1,IgG2a and IgG2b. Conclusion The highly effective and specific McAbs G11 can play an important role in diagnosis, treatment and prevention of the infection of AIV_H5N1