The hybrid peptide CA(1–7)-T(4–19) gene was designed according to the N-terminal 1–7 amino acid sequence of the antimicrobial peptide cecropinA (CA) and the N-terminal 4–19 amino acid sequence of thanatin (T), and synthesized by using Pichia pastoris preferred codons. Pichia pastoris hybrid expression vector was constructed, transformed into the prepared expression strain X-33 competent cells, and regulated by the alcohol oxidase (AOX). The biological activity studies after purification have shown that the hybrid antimicrobial peptides were equipped with broad-spectrum antimicrobial activity, and had a good antimicrobial effect on the majority of Gram-positive and Gram-negative bacteria without hemolysis and good stability in vitro as well. Minimum inhibitory concentration (MIC) of the hybrid peptide on common resistant pathogens in clinics was obtained. It provided a new field for researchers to find ideal antimicrobial agents. Therefore, the antimicrobial peptides have great potential for application. Keywords: Hybrid antimicrobial peptide, CecropinA, Thanatin, Pichia expression system, Antibacterial activity