The invention discloses a preparation method of a beta-defensin 2 antibacterial peptide. The preparation method comprises the following steps: S1, selecting Fmoc-Pro-2-Chloro-Resin resin, carrying out swelling, and then removing an Fmoc protecting group; and S2, according to the sequence of the beta-defensin 2 antibacterial peptide, sequentially carrying out coupling from the C terminal to the N terminal until the last amino acid Fmoc-Gly-OH, and removing the Fmoc protecting group to obtain the beta-defensin 2 antibacterial peptide resin peptide. According to the preparation method, an Fmoc solid phase method is used, short dipeptide and short tripeptide are selected as reaction raw materials for coupling, generation of impurities is reduced, the idea that a pair of disulfide bonds is formed through oxidation to form a relatively stable structure, and then the remaining two pairs of disulfide bonds are formed through natural oxidation is adopted, so that the purposes of high purity and high yield of a crude product are achieved; the method has the advantages of high operability and reduction of emission of three wastes, can be used for large-scale production, has high theoretical guidance value and considerable economic and practical value, and is wide in application prospect.