The invention discloses an MLH fusion antibacterial peptide, a preparation method and application thereof. Housefly antibacterial peptide, LL-37 and helicobacter pylori peptide are adopted as the parent peptide, a bioinformatics method is utilized for analysis to obtain an amino acid sequence of MLH, and a gene fragment of the hybrid gene MLH is encoded according to the E.coli codon preference. SOE-PCR technology is employed to synthesize a required target gene, the target gene is connected to the expression vector pET-32a(+), and the obtained recombinant expression vector is transferred into an escherichia coli expression strain to construct the recombinant genetic engineering strain pET-32a-MLH/BL21(DE3), IPTG induced protein fusion expression is carried out, and finally bacteriostatic activity identification is conducted on the obtained fusion expressed hybrid peptide, and the result indicates that the fusion expressed hybrid peptide has significant bacteriostatic effect on staphylococcus aureus and bacillus subtilis.