Bacillus velezensis is a novel species in the Bacillus genus which exhibits a broad spectrum of antimicrobial activities because it produces various secondary metabolites. The current research on B. velezensis mainly focuses on its use for the promotion of the growth of animals and plants, for antagonizing pathogens, and on investigating its gene cluster which plays significant roles in biological control, drug research and development, and food fermentation. The aims of this study are to explore the gene clusters related to antagonistic substances of B. velezensis LF01 strain based on the whole genome sequencing, and to evaluate the biological safety and biocontrol effect of the antagonistic substances of LF01 strain. In this study, the whole genome of LF01 strain was sequenced based on the third generation Nanopore sequencing platform, and its taxonomic status was identified based on ANI and DDH online analysis, and the genetic evolution at genome level. The gene clusters related to antagonistic substances of LF01 strain was analyzed based on antiSMASH software. The hydrolysis activity of LF01 strain to carbohydrate was analyzed based on CAZyme database. The toxicity of the antagonistic substances of LF01 strain to Oreochromis niloticus and Danio rerio was evaluated by intraperitoneal injection. The biocontrol effect of the antagonistic substances of LF01 strain to O. niloticus was evaluated by artificial infection after 5 d of feeding O. niloticus. The complete genome of LF01 strain had a total length of 3974023 bp and the GC content was 46.56%, and it contains 3843 coding genes. Compared to B. velezensis, the ANI and DDH values of LF01 strain were ≥97.66% and ≥80.10%, respectively. Furthermore, the genomic evolution analysis showed that LF01 strain was clustered with B. velezensis. These results indicated that LF01 strain was identified as B. velezensis at the genomic level. The LF01 strain contains nine gene clusters related to antagonistic substances, including bacillaene, bacillibactin, bacillomycin D, bacilysin, difficidin, fengycin, macrolactin H, plantazolicin and surfactin, which accounts for 8.83% of the total genome sequences. In addition, LF01 strain contains a large number of CAZymes related to the degradation of cellulose, hemicellulose, starch, chitin, pectin, peptidoglycan and glucan. The antagonistic substances of the LF01 strain showed high biological safety to O. niloticus and D. rerio, and they significantly enhanced the resistance of O. niloticus against S. agalactiae infection. In conclusion, LF01 strain contains a large number of gene clusters for biosynthesis of secondary metabolites related to antagonistic substances. The antagonistic substances were found to be safe for O. niloticus and D. rerio, and feeding O. niloticus with them significantly improved the diseases resistance of fish. Therefore, the antagonistic substances of LF01 strain have a broad development and application prospect as biocontrol agents to improve bacteria diseases control in aquaculture.