Objective of this paper is to excavate the siderophore synthesis gene from Brevibacillus brevis GZDF3 and verify its type and antibacterial effects. The method is using genome mining technology to analyze the siderophore synthesis genes and the phylogenetic tree of each synthesis gene was constructed separately. Iron free medium was utilized to induce the synthesis of siderophore and CAS liquid detection method was used for qualitative and quantitative analysis on siderophore. The type of siderophore was preliminaries identified by Arnow and its antibacterial effects were analyzed according to the agar punching method. The results show that a siderophore synthesis gene cluster with 83% similarity to Petrobactin was found in Brevibacillus brevis GZDF3 genome. Iron free medium could induce siderophore synthesis and the optimal incubation time cultured in iron free medium was 30 h and 48 h. Antagonistic strain GZDF3 had the capacity to synthesize catechol-type siderophore. Also, GZDF3 had a powerful antibacterial effect on pathogenic fungus Fusarium oxysporum of rotted root on Pinellia ternata. Therefore, Brevibacillus brevis GZDF3 can produce catechol-type siderophore in an iron-deficient culture medium, which was also a main antifungal active substance.