Abstract: Grouper nervous necrosis virus（GNNV）infects the nervous system of juvenile fish, with mortality rates as high as 90%–100%, posing a major bottleneck to the sustainable development of grouper aquaculture. The preparation of polyclonal antibodies against GNNV is essential for in-depth study of viral protein functions and the development of related detection methods. In this study, an expression vector containing the protruding domain（P-domain）of the GNNV capsid protein, designated GNNV-P-pET-15b, was constructed and transformed into Escherichia coli BL21（DE3）for prokaryotic expression. The purified GNNV-P protein was used as an antigen to immunize New Zealand white rabbits, generating rabbit-derived polyclonal antiserum. The antiserum titer was determined by indirect ELISA, and its specificity was analyzed by Western blot. Results showed that a soluble recombinant GNNV-P protein of approximately 15 kDa was successfully obtained via prokaryotic expression. Immunization with this antigen produced GNNV-P-specific rabbit polyclonal antiserum with a titer exceeding 1∶150,000 as measured by indirect ELISA. Western blot analysis confirmed the high specificity of the antibody. These findings provide key biological materials for further research into the infection mechanism of GNNV and the development of related immunological detection methods.