Abstract: Red spotted grouper nervous necrosis virus (RGNNV) is one of the most significant pathogens threatening marine fish farming in China, so early detection of RGNNV is a critical measure for preventing viral infection and reducing aquaculture losses. To develop a rapid RGNNV detection method, we developed a rapid visual detection method by integrating recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system. Besides, we designed specific RPA primers and crRNA, then optimized both RPA reaction conditions and CRISPR/Cas12a detection parameters. The method's sensitivity and specificity were systematically evaluated through clinical testing of 20 suspected-infected grouper samples, with comparison to the standardized qPCR method (SC/T 7216—2022). Results show a detection limit of 1.5×10¹ copies·µL⁻¹ and complete concordance with qPCR findings. The established RPA-CRISPR/Cas12a method demonstrates rapid detection, high sensitivity, excellent specificity, and visual interpretability, providing an efficient field-deployable solution for RGNNV surveillance in aquaculture operation.