Abstract: The thermostable β-galactosidase-producing strain Bacillus stearothermophilus XG24 was used as an original strain which was induced by microwave and NTG for increasing the β-galactosidase production ability. One of the highest efficient β-galactosidase-producing mutants was named as XGN52 with its stable β-galactosidase production ability. Its cumulative β-galactosidase yield was 31.59U/mL, 115.92% higher than that of the original strain. Varieties of parameters of β-galactosidase production fermentation were studied, including fermentation time, initial pH, incubation temperature. The result indicated the optimum condition of β-galactosidase production for the mutant XGN52 as fermentation time 36h, initial pH7.0 and temperature 42℃.The β-galactosidase production characters of the mutant and the original strain were different. This work showed a good application potential of microwave-NTG combined mutation in the β-galactosidase production.