The development of antibiotic-resistant bacteria has become a major public health problem, prompting the search for alternative solutions. Tachyplesin I (TP-I) is an antimicrobial peptide, which exhibits potent and broad-spectrum activities against bacteria, fungi, viruses, and tumor cells. However, limited amounts of TP-I produced in horseshoe crab restrict its large-scale use. In order to solve this problem, a eukaryotic expression system of Pichia pastoris with high TP-I expression was constructed by gene engineering. To achieve high expression of TP-I, 74 amino acid-long peptide (4TP-1) was designed containing 4 copies of TP-I, and specific cleavage sites for pancreatic elastase (-Ala down arrow or -Gly down arrow) and carboxypeptidase A (cleaves C terminal amino acid); these cleavage sites for enzymes were located between the four copies of TP-I. The gene sequence for the designed peptide was synthesized taking into consideration codon preferences for P. pastoris, and cloned into the highly efficient expression vector pGAPZ alpha B. Host Pichia pastoris strain GS115 cells were transfected by the constructed expression vector pGAPZ alpha B-4tp-I by electroporation. Tricine-SDS-PAGE electrophoresis was carried out to detect the expression of target peptides in the fermentation medium. This analysis showed a protein band of 3.3 kDa, identical to that of chemically synthesized TP-I, verifying that successful synthesis and secretion of TP-I by genetically engineered R pastoris. The concentration of TP-I in the fermentation broth was 27.24-29.53 mg/L. High-resolution mass spectrometry analysis documented that the TP-I monomer had the same molecular weight, 2262.85, as the designed 17-amino acid sequence. The recombinant TP-I peptide displayed different levels of bactericidal activity against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Thus, the present study demonstrated the feasibility of achieving high levels of expression of TP-I in P. pastoris.