The application provides polypeptide Pv26-3 with antimicrobial activity, belonging to the technical field of genetic engineering drugs. In the polypeptide Pv26-3, tryptophan W replaces the 7th, 11th,21st and 25th sites in the amino acid sequence of a parent molecule Pv26, and lysine K replaces amino acids at the 1st, 12th, 20th and 24th sites, thereby enhancing the hydrophobicity and positive charge of the parent molecule Pv26. Since a hydrophobic group plays a key role in the process of inserting antimicrobial peptide into a cell membrane, the improvement of hydrophobicity is beneficial to the antimicrobial effect of the antimicrobial peptide; the antimicrobial peptide is bonded to the membrane by electrostatic interaction, so as to promote the combination of the hydrophobic group into the membrane, and then the membrane structure is destroyed to cause cell death, so that positive charge is the key factor affecting the binding of the antimicrobial peptide and the membrane; by improving hydrophobicity and positive charge, the binding ability of the antimicrobial peptide and the membrane can be enhanced, the antimicrobial activity and antibacterial effect of antimicrobial peptide can be improved, and at the same time, the action concentration of antimicrobial peptide is lowered.