Reactive oxygen species (ROS) serve as signal molecules in plant defense responses, and the respiratory burst oxidase homolog (RBOH) enzyme plays a crucial role in their production. Although numerous RBOH family members have been identified in various plants, little is known about the RBOH genes in blueberries. In this study, we identified six VcRBOH genes from the blueberry genome. Phylogenetic analysis revealed that these VcRBOH genes can be classified into three subgroups. Conserved domain and motif analysis demonstrated high sequence similarity among VcRBOH proteins. Analysis of cis-acting elements suggested that VcRBOH genes may be involved in stress, light, and phytohormone responsiveness. Based on transcriptome data, we observed low expression levels of VcRBOHB, VcRBOHC, and VcRBOHE during the flower_at_anthesis stage. In contrast, VcRBOHA and VcRBOHD showed relatively high expression levels in various tissues. The reverse-transcription quantitative PCR (RT-qPCR) analysis indicated rapid induction of VcRBOHF by flg22 and chitin treatments. Notably, overexpression of VcRBOHF in Arabidopsis promoted PTI responses, including increased expression of marker genes, ROS production, and callose deposition. Moreover, the overexpression of VcRBOHF resulted in enhanced disease resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 infection. These findings provide valuable insights into the roles of VcRBOHF genes in plant defense responses and lay the groundwork for a more comprehensive understanding of the molecular mechanisms underpinning blueberry disease resistance.