The invention discloses novel antimicrobial peptide Hainanenin-5 of Amolops hainanensis, a gene of the antimicrobial peptide Hainanenin-5 of Amolops hainanensis, a separation method and a chemical synthesis method for the antimicrobial peptide Hainanenin-5 of Amolops hainanensis and application of the gene of the antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and belongs to the technical field of biomedicine. The Hainanenin-5 is small peptide which is obtained by the steps of electrically stimulating the Amolops hainanensis so as to collect a skin exudate, centrifuging, freeze-drying, performing gel filtration column chromatography and reverse phase-high performance liquid chromatography (RP-HPLC) and purifying, and has a C terminal containing a seven-membered ring; the molecular weight is 2,337.91; and the isoelectric point is 10.11. A primary structure of the Hainanenin-5 is Phe<1>a<2>Leu<3>Gly<4>Ala<5>Val<6>Thr<7>Lys<8>Arg<9>Leu<10>Pro<11>Ser<12>Leu<13>Phe<14>Cys<15>Leu<16>Ile<17>Thr<18>Arg<19>Lys<20>Cys<21>. A gene for encoding a Hainanenin-5 precursor consists of 321 nucleotides, wherein a mature peptide part is encoded by 139th to 201st nucleotides. The Hainanenin-5 has low molecular weight and a simple structure, only contains one disulfide bond, and can be conveniently prepared through chemical synthesis and genetic engineering. The Hainanenin-5 is used for preparing a therapeutic medicine for pathogenic microorganism infectious diseases, has broad-spectrum antimicrobial activity, has the activities on gram-positive bacteria, gram-negative bacteria and fungi (including clinically separated medicine-resistant strains), and also has the advantages of low hemolysis.