The invention discloses an oil emulsification turbot vaccine, which is prepared by mixing vaccine antigen Vibrio anguillarum O1 serotype strain VAM003 and O2 serotype strain VAM007 and emulsifying themixture with Marcol 52. The invention further discloses an application of the oil emulsification turbot vaccine at the same time, the final concentration for immunizing the turbot vaccine is 109 cells/ml, and the concentration for injection immunization is 104-108 CFU/g. The vaccine has the advantages that the immune turbot can obtain higher antibody titer and early immune protection effect; andthe vaccine has extremely small side effects, and has no significant influence on the weight of turbot in production scale.