Bacillus subtilis is widely used for the large-scale industrial production of proteins. In this study, the transcriptomes of B. subtilis 168 and B. megaterium DSM319 cells grown in stationary phase were analyzed to expand the repertoire of highly-active promoters for high-level protein expression based on the transcriptomes of these Bacillus strains. 24 genes with the highest expression levels among 2048 highly expressed gene families were chosen to examine promoter activity. The activities of four promoters with the beta-galactosidase (bgaB) gene as a reporter were stronger than those of the well-characterized strong promoter P43. The expression level of recombinant Pro-transglutaminase (pro-MTG) from Streptomyces mobaraensis achieved 87.6 U/mL and 70.7 U/mL wider the control of two constitutive promoter P(s)(odA)( )and P-yd(zA), respectively, compared to the promoter P43. Our study provides a basis for further studies on the Bacillus transcriptome by identifying strong promoters for industrial uses.