Abstract: Siderophore is considered as one of the virulence factors of Aeromonas hydrophila and is encoded by seven genes of amoCEBFAGH. AmoCGH have been confirmed to be involved in the synthesis of siderophore in previous studies. RT-PCR assay indicated that the expression of amoAEF genes were regulated by iron. In order to further explore the function of genes amoAEF, in this study, we used the suicide plasmid PRE112 to construct gene deletion mutant strains ΔamoA, ΔamoE and ΔamoF based on the infusion PCR and gene homologous recombination principle. The detection of the siderophore synthesis in the wild strains and mutant strains were performed using CAS plate test and arnow test. The results showed that Aeromonas hydrophila ΔamoA, ΔamoE and ΔamoF were successfully constructed. The growth of wild strain, ΔamoA, ΔamoE and ΔamoF had no significant difference in iron-riched medium, while in iron-depleted medium, the growth and siderophore synthesis capacity of ΔamoA, ΔamoE and ΔamoF were significantly lower than that of the wild strain. The results suggested that amoA, amoE and amoF are the critical synthesis genes of siderophore, and the growth of the deletion mutants were inhibited under low iron conditions.