Abstract: Objective: To reveal the bacteriostatic activity of hybrid antimicrobial peptide NK-LPd, and discuss its further exploitation potential. Methods: Two glycines were utilized as the linker to connect the active fragments of NK-lysin and Piscidin, and the physicochemical properties and functional structures of the hybrid antimicrobial peptides were predicted by bioinformatics. The hybrid antimicrobial peptide NK-LPd gene was optimized according to Pichia pastoris codon preference principle. The optimized gene was amplified by gene splicing via overlap extension polymerase chain reaction (gene splicing by overlap extension polymerase chain reaction, SOE-PCR) and inserted into the secretory expression vector pPIC9K. The recombinant plasmid was chemically transformed into P. pastoris KM71 competent cells. The recombinant expression was induced by 0.5% methanol as the inducer, which products were purified by affinity purification and its activity was evaluated. Results: KM71/pPIC9K-NK-LPd was successfully constructed. After expression for 5 days, an expression product with a relative molecular mass of about 6 kDa was obtained in the supernatant, which was in line with the expected size. Bacteriostatic activity experiments showed that NK-LPd had strong bacteriostatic activity against Gram-negative and Gram-positive bacteria. Compared with the parent peptides, the bacteriostatic activity of NK-LPd was significantly enhanced. Conclusion: The hybrid antimicrobial peptide NK-LPd was designed and expressed in P. pastoris KM71, and its bacteriostatic activity was superior to that of the parent peptides. This study could provide technical reference for the design and production of novel hybrid antimicrobial peptides.