Human lysozyme (hLYZ) is widely used in food preservation, animal feed, biomedicine for its antibacterial properties. In this paper,lysozyme gene codon was optimized according to the codon bias of Pichia pastoris. Then pPIC9K-hlyz was transformed into GS115 chromosome through homologous recombination. Recombinant Pichia pastoris G-P-hlyz was successfully screened by 6.0 mg·mL^-1 G418 and colony PCR. At 28 ℃, pH 6.0, 240 r·min^-1,1% methanol, induced expression for 120 h, the recombinant protein was secretly expressed, the hLYZ activity of the fermentation broth reached (2 414.9±108.1) U·mL^-1, and the protein content was 166.7 μg·mL^-1. After the supermatant of fermentation broth was purified by Sephadex G50, hLYZ was purified as a single protein with a molecular weight of approximately 14.7 kDa which is verified by the SDS-PAGE analysis results. In addition, in vitro antibacterial experiments, hLYZ has better antibacterial effect on gram-positive bacteria than that on gram-negative bacteria. More importantly, the antibacterial activity is 10-20 times higher than the standard product.