The invention relates to human alpha-defensin 5 (HD5) antiviral active mutant polypeptide and preparation method and application. The 21th glutamic acid in HD5 of the amino acid sequence of the mutant polypeptide can be replaced into arginine (E21R). The preparation method of the mutant polypeptide has two preparation methods, namely a solid-phase chemical synthesis method and a gene engineering preparation method. The HD5 mutant polypeptide of the invention has efficient antiviral activity under the conditions that serum exits or absents, and compared with parent HD5 polypeptide, the antiviral function of the invention is obviously improved. The HD5 mutant polypeptide of the invention is used for preventing viral infection, such as HSV, HPV, HIV and the like.