To study the role of L protein in the snakehead vesiculovirus (SHVV) proliferation,the first 900 bases of the SHVV L gene was amplified and inserted into pET-32a (+) to construct pET32a-L prokaryotic expression plasmid,and transformed into E. coli BL21 (DE3) competent cells. Different temperature,IPTG concentration and induction time were designed and the best expression conditions were selected. The purified protein was purified by NI-NTA affinity chromatography column,and was used to immunize New Zealand white rabbits to prepare polyclonal antibodies. Western blot was used to identify the specificity of the antibody.Indirect immunofluorescence was used to study the location of L protein in channel catfish ovary (CCO) cells after SHVV infection. The results showed that the purified L protein was about 42 ku. The L protein polyclonal antibody can react specifically with the L protein,and the L protein is mainly localized in cytoplasm,indicating that the L protein polyclonal antibody has been successfully prepared.