Bacillus subtilis, a key model strain in microbial physiology and biochemistry research, is non-pathogenic, secretes proteins efficiently, and has a clear genetic background, making it an ideal host for producing heterologous proteins, such as small molecules, bulk chemicals, enzymes, and functional peptides. In recent years, the synthetic biotechnology and metabolic engineering methods for B. subtilis have advanced rapidly, providing excellent tools and theoretical references for producing target products using this chassis cell. This review systematically summarizes strain optimization strategies via screening and modification of wild-type B. subtilis strains as expression hosts, promoter engineering, fine-tuning gene expression based on proteases and molecular chaperones, plasmid-based expression systems regulation, as well as its applications. Detailed analysis was conducted from a novel perspective on the comparison between B. subtilis and E. coli as hosts for biological production. Furthermore, the review delves into important considerations and future research directions for optimizing B. subtilis expression systems in industrial production. With the development of gene editing and synthetic biology technologies, and the continuous optimization of strain improvement strategies, B. subtilis will demonstrate tremendous potentials as a major microbial cell factory for the industrial production of protein products.