This study is pertaining to the production of penicillin G acylase (PGA) by Bacillus subtilis 8105MU331 in which PGA gene, under the control of thermal-induced promoter, was integrated. The key process parameters including induced-temperature, induced- time, and culture temperature were optimized in flask culture. A three-stage cultivation process was developed for PGA production with the expression system of B. subtilis 8105MU331. Furthermore, a bioreactor with a thermal-induced apparatus was designed for continuous production of PGA, where cell growth, induction, and PGA expression could be conducted separately. At a dilution rate of 0.20 h–1, PGA production was taken under continuous cultivation in three-stage process. After continuous feeding, the cell density, pH, and residual glucose in the first- and third-reactor were maintained steady for up to 40 h. These results suggested that the new three-stage process might be feasible and very efficient for production of heterologous proteins.