Bacillus subtilis is a well-characterized Gram-positive bacterium and a valuable host for recombinant protein production because of its efficient secretion ability, high yield, and non-toxicity. Here, we comprehensively review the recent studies on recombinant protein production in B. subtilis to update and supplement other previous reviews. We have focused on several aspects, including optimization of B. subtilis strains, enhancement and regulation of expression, improvement of secretion level, surface display of proteins, and fermentation optimization. Among them, optimization of B. subtilis strains mainly involves undirected chemical/physical mutagenesis and selection and genetic manipulation; enhancement and regulation of expression comprises autonomous plasmid and integrated expression, promoter regulation and engineering, and fine-tuning gene expression based on proteases and molecular chaperones; improvement of secretion level predominantly involves secretion pathway and signal peptide screening and optimization; surface display of proteins includes surface display of proteins on spores or vegetative cells; and fermentation optimization incorporates medium optimization, process condition optimization, and feeding strategy optimization. Furthermore, we propose some novel methods and future challenges for recombinant protein production in B. subtilis.