Abstract: In this study, a high-yield aminopeptidase producing strain, Bacillus subtilis LBJ4-5, was isolated from Pixian Douban, and its safety was evaluated through hemolysis and antibiotic sensitivity tests. Additionally, Illumina second-generation sequencing and PacBio third-generation high-throughput sequencing were employed for the whole-genome sequencing of B. subtilis LBJ4-5 to characterize its genomic features. Gene prediction and functional annotation were carried out using databases such as COG, GO, KEGG, CAZY, CARD and VFDB. Furthermore, the aminopeptidase production capacity and safety of the strain were evaluated.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 was highly sensitive (diameter≥20 mm) to 10 antibiotics, including cefazolin, vancomycin, penicillin G, and cefadroxil, indicating a favorable safety profile.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 COG, GO, KEGG, CAZY, CARD, and VFDB databases identified 3386, 2581, 2552, 144, 256 and 401 functional genes, respectively. Further analysis indicated that the genome of B. subtilis LBJ4-5 contains 31 aminopeptidase-encoding genes. Additionally, the prediction of virulence and antibiotic resistance genes further confirmed the safety of the B. subtilis LBJ4-5 strain. In conclusion, B. subtilis LBJ4-5 can be used as a fermentation starter for the processing of fermented foods.