Abstract: The low yield of natural Bovine Lactoferricin peptide (LfcinB) and the high cost associated with its chemical synthesis made genetic engineering the preferred method for its production. This study used bioinformatics approaches to analyze the fundamental physicochemical properties of fusion proteins. By conjugating the anionic peptide antioxidant peptide from bullfrog skin protein (APBSP) with LfcinB through a fusion protein expression strategy, we successfully neutralized the cationic charges carried by LfcinB. The recombinant yeast strain GS115/pPIC9K-EGFP-APBSP-LfcinB-His was constructed and subsequently screened for optimal expression levels. The expressed product LfcinB-His underwent systematic purification, characterization, and antibacterial activity evaluation. The experimental data revealed that while native LfcinB retained potent antimicrobial activity, its engineered recombinant variants, APBSP-LfcinB and EGFP-APBSP-LfcinB-His, exhibited no detectable antibacterial properties. Under methanol induction, the recombinant Pichia pastoris strain GS115/pPIC9K-EGFP-APBSP-LfcinB-His demonstrated a marked growth advantage over GS115/pPIC9K-LfcinB-His. SDS-PAGE analysis confirmed high-efficiency expression of the fusion protein. Purified LfcinB-His solution exhibited substantial antimicrobial potency against four bacterial strains: Staphylococcus aureus ATCC25923 (3.62×10⁹ U/mL), Staphylococcus aureus CMCC26003 (1.98×10⁸ U/mL), Escherichia coli ATCC25922 (1.86×10⁸ U/mL), and Escherichia coli C600 (1.54×10⁹ U/mL). This study lays the foundation for the further development of high-density fermentation for the production of LfcinB.