The invention relates to an aquaculture animal disease prevention and control technology, specifically to a Vibrio anguillarum 01 serotype inactivated vaccine, a preparation method and a use method thereof, wherein the vaccine is Vibrio anguillarum 01 serotype, the Vibrio anguillarum 01 serotype strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 7, 2012, and a preservation number is CGMCC No.6942. With the inactivated vaccine, breeding fishes can be effectively stimulated to produce specific immune response, immunity of breeding animals can be effectively enhanced, and susceptible fishes can be protected from pathogenic Vibrio anguillarum infection. In addition, the Vibrio anguillarum 01 serotype inactivated vaccine preparation method has characteristics of low raw material cost and safety on animals and environment.