Viral hemorrhagic septicemia virus (VHSV) is one of the most serious pathogens of finfish that affects over 80 marine and freshwater species in North America, Europe, and Asia. The genome of VHSV is a negative-sense, single stranded RNA containing approximately 12000 base pairs that encode six proteins, which are the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV), and RNA polymerase protein (L) in order from 3' to 5'. The M protein functions in a variety of rhabdovirus infection processes such as assembly and budding, inhibiting host-cell directed transcription and gene expression, inducing apoptosis of the host cell, et al., which were all thoroughly investigated in other rhabdoviruses such as vesicular stomatitis virus and rabies virus, while less so in VHSV. In order to investigate the function of the M protein of VHSV, the M gene was amplified by PCR and cloned into the prokaryotic expression vector pET-32a(+), which was transformed into Rosetta (DE3) competent cells. The recombinant protein was induced by IPTG, and the polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant protein. The titer of the antibody was detected by an indirect ELISA, and the antibody specificity was tested by western blot and indirect immunofluorescence. The results showed that the full-length M gene was 606 bp. The fusion protein induced by IPTG mainly existed in the form of an inclusion body, and the size was about 36 kDa, which was slightly smaller than expected. The indirect ELISA assay showed that the titer of the antibody was greater than 1:102400. The western blot showed that the antibody could specifically identify the purified fusion protein and M protein in VHSV infected (EPC) cells. The indirect immunofluorescence showed that the M protein antibody can recognize the M protein in VHSV infected EPC cells, and the M protein was mainly localized in the cytoplasm and cell membrane. These results suggest that the prepared polyclonal antibody can be used as an effective tool to study the function of the M protein and for the diagnosis of VHSV.