Abstract: In order to express a broad-spectrum bacteriostatic protein with high efficiency and low toxicity, epinephelus coioides melittin（MEL） gene of grouper and c type lysozyme（Ec-cLYZ） gene were linked via T2A and cloned into the vector pPICZαA to construct the recombinant plamids pPICZαA-Ec-cLYZ-MEL. The correctly identified recombinant expression plasmid was linearized by restriction endonuclease SacⅠ and was electrotransformed into receptive cells of Pichia pastoris GS115 to construct Pichia pastoris expression strains. The recombinant strains were induced by 0.5% methanol. The supernatant was collected for freeze-drying, concentration and purification. The expression of the recombinant protein was verified by Western-blot. The protein concentration was determined by BCA method. Hemolytic activity of different concentrations of recombinant protein on rabbit erythrocytes was detected by test tube double dilution method. The antibacterial activity of the recombinant protein was evaluated by Oxford cup method. The results showed that the recombinant plasmids pPICZαA-Ec-cLYZ-MEL, pPICZαA-Ec-cLYZ and pPICZαA-MEL were successfully constructed; the expression strains GS115/pPICZαA-Ec-cLYZ-MEL, GS115/pPICZαA-Ec-cLYZ and GS115/pPICZαA-MEL. After induction, GS115/pPICZαA-Ec-cLYZ-MEL expressed protein with molecular masses of 16.5, 3.4 ku, GS115/pPICZαA-Ec-cLYZ expressed protein with molecular masses of 16.5 ku, and GS115/pPICZαA-MEL expressed protein with molecular masses of 3.4 ku. The concentrations of recombinant protein Ec-cLYZ-MEL, Ec-cLYZ, and MEL in the supernatant were 35.49, 28.73, 27.31 mg/L, respectively. The hemolysis of rabbit erythrocytes by recombinant protein Ec-cLYZ-MEL, Ec-cLYZ, and MEL treated at the concentration of 150 mg/L was 2.3%, 2.9%, and 79.0%, respectively.Streptococcus alactiae was highly sensitive to recombinant protein Ec-cLYZ-MEL,Staphylococcus epidermidis moderately sensitive to recombinant protein Ec-cLYZ-MEL,Staphylococcus aureus and Escherichia coli were moderately sensitive to recombinant protein Ec-cLYZ-MEL, and recombinant protein Ec-cLYZ-MEL had better antibacterial zone effects than recombinant protein Ec-cLYZ-MEL. The results indicated that the recombinant protein Ec-cLYZ-MEL had no hemolytic activity on erythrocytes and had good bacteriostatic activity.