Abstract: To explore the relationship between the concentration of reactive oxygen and nitrogen species (RONS) in plasma activated water (PAW) and the activation parameters, as well as its effect on the inactivation of Escherichia coli, a plasma generator with a needle electrode structure is used to prepare PAW by dielectric barrier discharge in water. A series of PAWs is prepared under different conditions by adjusting different activation parameters, including plasma activation time and solution volume. Subsequently, the concentrations of HNO₃, H₂O₂, and O₃ in the PAWs, as well as the pH value, are analyzed. The variation of RONS is analyzed. The biological activity of the PAW is evaluated using sterilization experiments, and its inactivation of Escherichia coli is determined. The results show that increasing the discharge voltage, prolonging the activation time, or decreasing the solution volume can increase the concentration of RONS in PAW. The concentration of RONS is positively correlated with the biological activity of PAW, indicating that the higher the concentration of RONS, the better the bacterial inactivation effect of PAW. In particular, 10 mL of PAW prepared by activation for 3 min at 10 kV shows the highest biological activity and is able to achieve complete sterilization of Escherichia coli at concentration of 10⁵ CFU/mL. Finally, the antibacterial effect of PAW prepared by deionized water, normal saline, and tap water is compared. The results show that the antibacterial effect of PAW prepared by tap water is the best.