The invention relates to a vibrio anguillarum O3 serotype bacterial strain and an application method thereof. The vibrio anguillarum O3 serotype bacterial strain is separated from the body of scophthalmus maximu, is a wild bacterial strain with strong toxicity, and has the preservation number of CCTCC M 2016261. An antigen preparation method of the vibrio anguillarum O3 serotype bacterial strain comprises any one or more of inactivated bacteria, bacterial ghost ingredients, attenuated bacterial strains, antigen subunits and antigen gene expression products. The produced vaccine can be a single component of an antigen prepared by the bacterial strain, or a combined vaccine is produced by mixing of the antigen prepared by the bacterial strain and antigens of other bacteria, or a vaccine is produced by the antigen of the prepared single or combined vaccine and an adjuvant. The vaccination mode during vaccine application can adopt injection immunization, bath immunization or oral immunization.