Abstract: Grass carp hemorrhagic disease, caused by grass carp reovirus type II (GCRV-II), is the most devastating viral disease affecting grass carp aquaculture in China. GCRV-II is highly virulent and can rapidly spread, leading to substantial economic losses annually. Current detection of GCRV still heavily relies on laboratory-based molecular techniques, which are complex and fail to meet the urgent need for on-site rapid diagnosis. To address this, this study aims to develop a rapid detection kit for GCRV-II based on colloidal gold immunochromatography. Two monoclonal antibodies, VP35#8 and VP35#18, specifically recognizing two distinct epitopes of the outer capsid protein VP35 of GCRV-II, were utilized. The VP35#8 antibody was conjugated with 20 nm colloidal gold particles to prepare the gold-labeled pad, while the VP35#18 antibody and goat anti-mouse IgG were immobilized on the test line (T-line) and control line (C-line) of the nitrocellulose membrane, respectively, to assemble the test strip. The results demonstrated that the kit could successfully detect GCRV-II from diseased grass carp and rare minnow within 10 minutes. It showed excellent specificity with no cross-reactivity against Spring Viraemia of Carp Virus (SVCV), Cyprinid herpesvirus 3 (CyHV-3), and GCRV-I. The detection sensitivity reached 1.76×10² copies/μL, and the kit remained stable after storage at 4–25°C for 12 months. The results were consistent with that obtained by the national standard method (GB/T 36190-2018). In summary, this study successfully developed a user-friendly, rapid, and reliable on-site detection tool for GCRV-II, providing solid technical support for the early diagnosis and control of grass carp hemorrhagic disease, which is of great significance for safeguarding the sustainability of the aquaculture industry.