The invention relates to the biotechnology field, in particular to an RV (rabies virus) splitting solution and an application thereof. The RV splitting solution comprises a splitting solution A and a splitting solution B, wherein the splitting solution A is prepared from components as follows: N-sodium lauroyl sarcosinate, Triton X-100 and glycogen; the splitting solution B is prepared from components as follows: sodium chloride and EDTA (ethylene diamine tetraacetic acid). The RV splitting solution can effectively split RV particles, and extraction of virus outer membrane segments containing spikes and matrix protein is facilitated. According to the application of the splitting solution in preparation of an RV antigen, the application method is simple and rapid, complete splitting is realized, the splitting solution is easy to remove, and the obtained RV antigen is high in purity.