[Background] Subtilisin is an important industrial protease with many applications. [Objective] The aim of this study is to improve the subtilisin production by screening promoters and signal peptides, and optimization of fermentation medium. [Methods] Bacillus licheniformis BL10, constructed in our previous research, was served as the host strain for subtilisin production, and four promoters (PbacA, P43, PaprE and PsrfA) and four signal peptides (SPVpr, SPSacB, SPSacC and SPAprE) were screened to improve subtilisin production, and the medium for subtilisin production was further optimized. [Results] Based on our results, the expression ratios among these four promoters were PbacA>PaprE>P43>PsrfA, and the secretion efficiencies were SPAprE>SPSacC>SPSacB>SPVpr, respectively. Among these strains, BL10/pPbacA-aprE displayed with the highest subtilisin activity (275.21 U/mL), which was increased by 64% compared with that of BL10/pHY-aprE (167.98 U/mL). Furthermore, the fermentation medium was optimized, and the orthogonal test was further applied to enhance subtilisin production. The maximum subtilisin activity (747.37 U/mL) was obtained in the optimized medium with 40.0 g/L corn starch, 50.0 g/L soybean meal, 4.0 g/L (NH4)2SO4, 3.0 g/L K2HPO4, 1.0 g/L CaCO3, increased by 4.45-fold compared with that of the initial condition. [Conclusion] Collectively, this study provided an effective strategy for enhanced production of subtilisin.