Stress is unavoidable in the aquaculture environment. Upon exposure to stress, the host stress hormones profoundly affect the pathogenicity of bacteria. It has been demonstrated that the inter-kingdom communication between bacteria and their hosts is mediated by catecholamines hormones epinephrine (Epi)/ norepinephrine (NE), and transduced by QseBC two-component system (TCS). In bacteria, QseBC TCS is a widely used signal transduction mechanism that facilitates in eliciting an adaptive response to various environmental stimuli. It consists of a membrane-associated sensor histidine kinase (HK) QseC and a cytoplasmic response regulator (RR) QseB. In most cases, QseC senses and recognizes environmental signals, following activation via self-phosphorylation at a conserved histidine residue. The phosphoryl group is then transferred to a conserved aspartate residue of QseB. The activated response regulator then exerts its regulation on bacterial virulence. Aeromonas hydrophila is a common aquatic bacterium and can cause motile Aeromonad septicemia (MAS) in aquatic animals especially fish. Recently, QseC has been reported to regulate the virulence of A. hydrophila in response to NE. However, the specific role of QseB in NE-enhanced virulence of A. hydrophila remains poorly understood. In this study, we constructed a qseB gene deletion mutant of A. hydrophila NJ-35 (ΔqseB) and examined the virulence both in vitro and in vivo in the presence or absence of NE (100 μmol/L). The results showed that the deletion of qseB gene significantly reduced the growth-promoting effect of NE on A. hydrophila compared to the wild type strain NJ-35. Meanwhile, the biofilm formation ability of the ΔqseB mutant was remarkably lower than that of the wild type strain NJ-35 in the presence of NE, while no significant difference was observed when bacteria were cultured without NE. Also, the hemolytic activities of ΔqseB exhibited significant decrease compared to the wild type strain NJ-35 with NE treatment. However, there were no significant differences in the motility and lipase activity between the ΔqseB mutant and wild type strain NJ-35 with or without NE. Additionally, the ΔqseB mutant displayed a dramatically decreased virulence in the experimental infection of Oreochromisc oaureus. In conclusion, our data suggest that the biofilm formation and hemolytic activity enhanced by NE may be dependent on the phosphorylated QseB in A. hydrophila NJ-35, indicating that QseB plays an important role in the outbreak of hemorrhagic septicemia disease induced by fish stress. Our study further reveals the pathogenesis of A. hydrophila in response to NE and may provide a new theoretical basis for the research on communication between bacteria and their hosts.