The invention relates to an antimicrobial peptide Cm-CATH2, a preparation method thereof and an application. The Cm-CATH2 is a straight chain polypeptide containing 33 amino acid residues, the theoretical isoelectric point is 12.96, molecular weight is 4089.97Da, and net charge is +12. A gene of a precursor cathelicidin of the encoding antimicrobial peptide Cm-CATH2 comprises 486 nucleotides. The Cm-CATH2 has high antibacterial activity for Gram-negative bacteria, Gram-positive bacteria and fungi, comprises various clinical drug-resistant bacteria, is low in minimal inhibitory concentration and rapid and lasting in sterilizing effect, does not contain disulfide bonds and cyclic structures, has the advantages of low hemolysis, cell toxicity, difficulty in generating drug resistance and the like, can replace an antibiotic for preparing clinical drugs resisting pathogenic microorganism infection, diminishing inflammation and the like, is applied to additives in daily chemicals such as cosmetics, health care products, food and feed, and has an excellent application prospect. Chemical synthesis and gene engineering preparation are facilitated.