Abstract: 【Objective】 The study aimed to analyze the whole genome sequence of Bacillus velezensis W-1 and to explore its biocontrol mechanism against Zanthoxylum bungeanum root rot disease, providing a bioinformatics basis for its effective development and application, as well as theoretical support for its development as a biopesticide. 【Method】 The whole genome of strain W-1 was sequenced using the PacBio third-generation platform sequencing technology, and the gene function annotation and comparative genomics analysis were performed based on the sequencing results. Mycelia growth rate method was used to verify the effects of ethyl acetate extract of strain W-1 fermentation liquid on the mycelium morphology of Z. bungeanum root rot pathogen, electrical conductivity of the pathogen spore suspension and nucleic acid content. 【Result】 The total genome length of strain W-1 was 4166284 bp, with a GC content of 46.32%, encoding 4037 protein genes. A total of 2935, 3087, and 2185 genes were annotated in the GO, eggNOG and KEGG databases respectively. Fourteen secondary metabolite synthesis gene clusters were predicted in strain W-1, including 9 known gene clusters such as surfactin, butirosin A/butirosin B, plantazolicin, macrolactin H, bacillaene, fengycin, difficidin, bacillibactin and bacilysin, along with 5 unknown gene clusters. There were 53 unique gene families and 414 unique genes in strain W-1, showing close genetic relationship with the model strain B. velezensis FZB42. The antagonistic strain W-1 extract greatly inhibited the mycelial growth of the Z. bungeanum root rot pathogen, with a minimum inhibitory concentration（MIC） of 4.50 mg/mL and a minimum bactericidal concentration（MFC） of 9.00 mg/mL. The extract of strain W-1could cause the exosmosis of intracellular contents from the pathogen mycelia. Compared with the blank control group, the relative electrical conductivity of the pathogen spore suspension and nucleic acid content increased by 57.63% and 64.91% respectively after treatment with an extract concentration of 9.00 mg/mL for 10 h. 【Conclusion】 B. velezensis W-1can produce a variety of antibacterial substances. The ethyl acetate extract of its fermentation liquid can damage the integrity of the cell membrane of Z. bungeanum root rot pathogen, indicating its potential for development as a biopesticide and its promising application in the green prevention and control of Z. bungeanum root rot.