According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mil gene was reconstructed, synthesized and cloned into pPICZalpha-A to construct the recombinant expression vector pPICZa-A-CM. The pPICZalpha-A-CM was transformed into yeast host strain X-33. Under the control of the promoter AOX1 (alcohol oxidase 1), a approximately 1.9 kD cecA-mil protein was expressed with the high level of 245 microg/mL after optimized the requirements for the flask-shaking culture fermentation of the Pichia pastoris rX-33/pPICZalpha-A-CM. The hybrid antibacterial peptide had a broad spectrum antibacterial activity on both gram-positive and gram-negative bacteria, especially showed potent antibacterial activity against ampicillin-resistant and kanamycin- resistant bacteria, such as Staphylococcus aureus (cowan I) and pathogenic E. coli (O1) from chicken. In addition, the hybrid antibacterial peptide showed an extreme heat-stable and acid-stable characteristic. Based on these characteristics, the recombinant antibacterial peptide CecA-mil display application foreground in the field of antisepsis of food, prevention of disease, additives of animal feedstuff and so on.