Vibrio parahaemolyticus contamination, causes serious foodborne illness, and it has become a global health problem. As a disinfectant, aqueous ozone can effectively kill a number of bacteria, viruses, parasites and other microorganisms. In this study, three factors, namely, the aqueous ozone concentration, the exposure time, and the bacterial density, were analyzed by response surface methodology, and the aqueous ozone concentration was the most influential factor in the sterilization ratio. Under low aqueous ozone concentrations (less than 0.125 mg/L), the bacterial cell membranes remained intact, and the ozone was detoxified by intracellular antioxidant enzymes (e.g., superoxide dismutase and catalase). Under high aqueous ozone concentrations (more than 1 mg/L), cell membranes were damaged by the degree of peripheral electronegativity at the cell surface and the concentration of lactate dehydrogenase (LDH) released into the extracellular space, and the ultra-structures of the cells were confirmed by transmission electron microscopy (TEM). Aqueous ozone penetrated the cells through leaking membranes, inactivated the enzymes, inhibited almost all the genes, and degraded the genetic materials of gDNA and total RNA, which eventually lead to cell death.