The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to BmSPI39 mutant and application thereof. BmSPI39 is composed of the 25th to 98th positions in SEQ ID NO.1. The BmSPI39 mutant is the amino acid sequence of BmSPI39, as shown in SEQ ID NO.1, the alanine at the 56th position is mutated to arginine, Lysine, Serine, Threonine, Glutamine, Tyrosine, Methionine, Leucine, Aspartic Acid, Glutamic Acid, Histidine, Cysteine, Valine, Astragalus Paragine, Isoleucine, Phenylalanine, Tryptophan, Proline or Glycine. The BmSPI39 mutants of the present invention all have inhibitory activity on subtilisin and elastase, and when mutated to arginine or lysine, they also obtain trypsin inhibitory activity, and such mutants can be used to prepare trypsin inhibitors , the application prospect is good.