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The application of endophytic bacteria, particularly members of the genus Bacillus, offers a promising strategy for the biocontrol of plant fungal diseases, owing to their sustainability and ecological safety. Although multiple secondary metabolites that demonstrate antifungal capacity have been identified in diverse endophytic bacteria, the regulatory mechanisms of their biosynthesis remain largely unknown. To elucidate this, we sequenced the entire genome of Bacillus amyloliquefaciens GKT04, a strain isolated from banana root, which showed high inhibitory activity against Fusarium oxysporum f. sp. cubense race 4 (FOC4). The GKT04 genome consists of a circular chromosome and a circular plasmid, which harbors 4,087 protein‐coding genes and 113 RNA genes. Eight gene clusters that could potentially encode antifungal components were identified. We further applied RNA‐Seq analysis to survey genome‐wide changes in the gene expression of strain GKT04 during its inhibition of FOC4. In total, 575 upregulated and 242 downregulated genes enriched in several amino acid and carbohydrate metabolism pathways were identified. Specifically, gene clusters associated with difficidin, bacillibactin, and bacilysin were significantly upregulated, and their gene regulatory networks were constructed. Our work thereby provides insights into the genomic features and gene expression patterns of this B. amyloliquefaciens strain, which presents an excellent potential for the biocontrol of Fusarium wilt., The genome of Bacillus amyloliquefaciens GKT04, a strain that showed high inhibitory activity against Fusarium wilt, harbors 4,087 protein‐coding genes and 113 RNA genes, including eight gene clusters that potentially encode antifungal components. Among those 575 upregulated and 242 downregulated genes were identified from transcriptome during the inhibition of Fusarium oxysporum f. sp. cubense race 4 of GKT04. The gene clusters associated with difficidin, bacillibactin, and bacilysin were significantly upregulated, and the corresponding gene regulatory networks were constructed.