Abstract: The genomic characteristics and gene functional annotation of Bacillus subtilis LBJ4-5, an aminopeptidase-producing strain were studied to predict the presence of aminopeptidase-related genes, resistance, and virulence genes. This strain was isolated from Pixian Douban, and its aminopeptidase activity was measured using the LNA method. The safety of the strain was evaluated through hemolysis and antibiotic sensitivity tests. Simultaneously, using the Illumina second-generation sequencing technology and the third-generation high-throughput Pacbio sequencing platform, the whole genome of B. subtilis LBJ4-5 was sequenced to obtain its genomic characteristic. Gene prediction and functional annotations were performed using COG, GO, KEGG, CAZY, CARD, and VFDB databases. The results showed that after 48 hours of shake flask cultivation, the aminopeptidase activity in the fermentation supernatant of B. subtilis LBJ4-5 reached 179.23 U/mL. The strain showed no hemolytic activity and had high sensitivity to 10 antibiotics, including cefazolin, vancomycin, erythromycin, penicillin G, cephalosporin, streptomycin, amikacin, tetracycline, minocycline, and gentamicin (diameter≥20 mm). The genome of B. subtilis LBJ4-5 consisted of a circular closed DNA and a circular plasmid, with a size of 4053926 bp and a GC content of 43.66%. A total of 4285 protein-coding genes, 85 tRNA genes, 33 rRNA genes, and 119 sRNAs were predicted. Functional gene annotations in the COG, GO, KEGG, CAZY, CARD, and VFDB databases identified 3386, 2581, 2552, 144, 256 and 401 functional genes, respectively. Further analysis indicated the presence of 31 genes encoding aminopeptidases in the genome of B. subtilis LBJ4-5 predictive analysis of virulence revealed that B. subtilis LBJ4-5 possessed only one virulence factor. In conclusion, the experimental results and the predicted aminopeptidase gene information offer a preliminary explanation for the high activity of aminopeptidases in B. subtilis LBJ4-5. Moreover, safety evaluations and analysis of virulence and resistance gene carriage confirmed the safety of B. subtilis LBJ4-5. This strain would be served as a fermentation agent in fermented food processing, enriching the aminopeptidase-producing strain pool.