Viral nervous necrosis (VNN) is a major viral disease affecting the central nervous system of over 170 marine fish species, including grouper. It is caused by the nervous necrosis virus (NNV), a non-enveloped, positive-sense single-stranded RNA virus belonging to the Betanodavirus genus. Fish infected with NNV exhibit symptoms such as abnormal swimming behavior and anorexia, with mortality rates reaching up to 100%. Currently, no commercial vaccine is available for grouper VNN disease, presenting an urgent need for an effective solution in the grouper aquaculture industry. In this study, we aimed to develop an effective vaccine against VNN using NNV virus-like particles (VLPs) produced through a plant expression system. The coding sequence of the RGNNV capsid protein was subcloned into an expression vector, fused with a rubisco transit peptide, and expressed in Nicotiana benthamiana using agrobacterium transient transfection. The expressed recombinant protein exhibited VLP properties, as confirmed by SDS-PAGE, Western blot, and transmission electron microscopy. To assess the immunogenicity of the produced RGNNV VLPs, immunization trials were conducted on red-spotted groupers (Epinephelus akaara) and seven-banded groupers (Epinephelus septemfasciatus) with varying antigen dosages and booster vaccinations. The challenge results demonstrated that the RGNNV VLP vaccine provided protection against NNV infection in both grouper species, showing significant reductions in mortality rates compared to the unvaccinated control group. Additionally, antibody titration assays revealed antigen dose-dependent antibody production in vaccinated fish. In conclusion, the RGNNV VLP vaccine developed through the plant expression system poses promise for use in preventing VNN disease outbreaks in grouper aquaculture.