The present invention provides a duck tambosu virus genetic engineering subunit vaccine and a preparation method and an application thereof. A nucleic acid molecule having a sequence shown in SEQ ID NO:1 or a nucleic acid molecule identical to 95% or more of the nucleotide sequence shown in the SEQ ID NO:1 encodes a duck tambosu virus E2 protein, an immune composition comprising the duck tambosu virus E2 protein can be used to prepare the duck tambosu virus genetic engineering subunit vaccine, antigenicity, immunogenicity and functions of the vaccine are similar to natural proteins, and the vaccine is relatively high in expression level, strong in the immunogenicity, free of pathogenicity to ducks, and can also be prepared by large-scale serum-free suspension culture in a bioreactor, which greatly reduces a production cost of the vaccine.