The present application disclosed a method for preparing a sterile transgenic fish, comprising constructing antisense RNA expression vector of salmon-type gonadotropin-releasing hormone, introducing the recombinant DNA fragment into carp oosperm by microinjection, and screening the sterile transgenic fish by Polymerase Chain Reaction and radioimmunoassay, wherein the expression vector comprising a promoter of carp beta actin (β-actin) gene, a complementary DNA fragment of antisense salmon-type gonadotropin-releasing hormone (sGnRH) gene from carp with 323 bp as a target gene comprising sGnRH decapeptide, the coding region of gonadotropin-releasing hormone associated peptide and 3′ non-coding sequence, and 3′ flanking sequence of grass carp growth hormone gene as a stop sequence. The method of the present invention is easy and convenient for operation which provides a basis for providing a technical platform of general applicability for solving the hereditary and ecological safety problems of transgenic fishes.