The development of efficient, low-cost, and robust expression systems is important for the mass production of proteins and natural products in large amounts using cell factories. Glycerol is an ideal carbon source for large-scale fermentation due to its low cost and favorable maintenance of the fermentation process. Here, we used the antiterminator protein GlpP and its target promoter PglpD to construct a highly efficient glycerol-inducible expression system (GIES) in Bacillus subtilis. This system was able to express heterologous genes in an autoinducible manner based on the sequential utilization of glucose and glycerol under the regulation of carbon catabolite repression. In such a system, the concentration of glycerol regulated the strength of gene expression, and the concentration of glucose affected both the timing of induction and the strength of gene expression. By enhancing GlpP, the GIES was further strengthened for high-level intracellular expression of aspartase and secretory expression of nattokinase. High yields of nattokinase in a 5-L fermenter through batch and fed-batch fermentation demonstrated the potential to apply the GIES for large-scale enzyme production. Through the evolution of the -10 box of PglpD, mutants with gradient activities were obtained. In addition, hybrid glycerol-inducible promoters were successfully constructed by combining the constitutive promoters and the 5' untranslated region of PglpD. Collectively, this study developed a GIES to obtain high-value products from inexpensive glycerol. More importantly, the great potential of the pair of inherent terminator and antiterminator protein as a portable biological tool for various purposes in synthetic biology is proposed. IMPORTANCE In this study, a GIES was constructed in B. subtilis by employing the antiterminator protein GlpP and the GlpP-regulated promoter PglpD. Based on the sequential utilization of glucose and glycerol by B. subtilis, the GIES was able to express genes in an autoinducible manner. The amounts and ratio of glucose and glycerol can regulate the gene induction timing and expression strength. The GIES was further applied for high yields of nattokinase, and its robustness in production scale-up was confirmed in a 5-L fermenter. The high-level expression of heterologous proteins demonstrated the huge application potential of the GIES. Furthermore, mutants of PglpD with gradient activities and hybrid glycerol-inducible promoters were obtained through the evolution of the -10 box of PglpD and the combination of the constitutive promoters and the 5' untranslated region of PglpD, respectively. These results demonstrated the use of the antiterminator protein as a regulator for various purposes in synthetic biology.