Abstract: Objective: The gene cloning, protein expression, purification and activity analysis of a new type of Proteus vulgaris bacteriophage lyase Lys66 were performed. Methods: The whole gene sequence of bacteriophage was compared in the Genbank database. The gene sequence of lysase was excavated and cloned. The protein was expressed in Escherichia coli and was further purified to explore its antibacterial effect. Results: A gene sequence with high similarity to lyase was discovered through comparison, with a size of 393 bp. By using ExPAsy Bioinformatics Resource Portal, the lyase was predicted that its molecular weight was 15.20 kDa, the isoelectric point was 9.40, and it was composed of 130 amino acids. The whole optimized synthetic gene was constructed onto vector pET-32α to obtain the recombinant plasmid pET-32α-Lys66. The recombinant plasmid was transferred into competent cells of E. coli BL21 (DE3) to induce its expression. After purification and validation, 1.86 mg/mL Lys66 protein was obtained. The diameter of the bacteriostatic ring of Lys66 lyase on the plate was 19.30 mm. Thirteen Gram-negative bacteria out of 15 tested strains treated with chloroform showed lytic activity, with a wide host spectrum. When Lys66 (1.89 mg/mL) was used in combination with ethylene diamine tetraacetic acid (1 mmol/L), the OD_{600 nm} decreased by 0.61 after 2 h, indicating a good antibacterial effect. Conclusion: The recombinant lysase Lys66 expressed in this study had good antibacterial effects and could be used as a potential antibacterial agent.