The invention discloses a pseudorabies virus gE/gI double gene deleted vaccine strain, as well as a construction method and application thereof, and belongs to the technical field of medicine. The pseudorabies virus gE/gI double gene deleted vaccine strain is constructed by knocking out gE and gI two genes based on a pseudorabies virus BJ strain, wherein the pseudorabies virus BJ strain is named BJ, which is preserved in the China General Microbiological Culture Collection Center with the address of Institute of Micorbiology, Chinese Academy of Sciences No.1 Beichen West Road, Chaoyang District, Beijing, has the preservation number of CGMCC No.15911. Experiments prove that a pseudorabies inactivated vaccine immunizing a primiparous healthy pig to prevent pseudorabies has the advantages ofsafety, quick antibody generation, durable immunizing period and the like, can be strategically matched with corresponding DIVA (differentiating infected from vaccinated animals), and has a wide application prospect in the field of pseudorabies prevention and treatment.