The invention belongs to the technical field of biology and relates to a preparation method and application of a novel antimicrobial peptide Misgurin mutant. The technical scheme is as follows: introducing glutamine at the C tail end of the amino acid sequence of natural Misgurin so as to design a novel Misgurin mutant; selecting Pichia yeast biased codons so as to artificially synthesize a complete gene of the novel Misgurin mutant; constructing a Pichia yeast secretion type expression vector pPICZaA-M; transforming a host cell SMD1168 by a vector electroporation method; selecting to obtain high-efficiency expression strains; and under proper culture conditions, ensuring 100 microgram of recombinant novel Misgurin mutant is contained in per milliliter of expression supernatant. The obtained product has a good nematocidal activity on staphylococcus aureus, pig salmonella and pig pathogenic Escherichia coli K88, K99 and 987P, and has an obvious effect on prevention and treatment of clinical bacterial diseases.