A novel biosensor strain (Escherichia coli ALM403) that responded to N-acyl homoserine lactone (AHL) was constructed using a luxR-Plux cassette as a regulatory sequence and β-mannanase as a reporter gene. Dinitrosalicylic acid method was used to detect the response of the sensor strain to N-acyl homoserine lactone. By investigating the response to a range of concentrations of N-β-oxooctanoyl-L-homoserine lactone (OOHL), it was demonstrated that the expression of mannanase in E. coli ALM403 could be greatly enhanced by OOHL and resulted in an assayable phenotype. A high-throughput screening approach was developed to isolate AHL-degrading microorganisms, and a marine Halomonas sp. S66-4 showing a marked AHL-degrading ability was successfully isolated. In conclusion, the bioassay system provided a simple and efficient approach to isolate AHL-degrading bacteria.