Vaccination has been regarded as the most effective way to reduce death and morbidity caused by infectious diseases in the livestock industry. In this study, plasma activated water (PAW) was introduced to prepare the inactivated Newcastle disease vaccine. Humoral immune response was tested by hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). In addition, cell-mediated immune response was evaluated by lymphocyte proliferation assay and flow cytometry. The results demonstrated that the vaccine prepared by PAW at appropriate volume ratio could induce similar antibody titers in specific pathogen-free (SPF) chickens compared with the formaldehyde-inactivated vaccine. The challenge experiment further confirmed that the vaccine prepared by PAW conferred solid protection against virulent NDV. Moreover, it was found that the vaccine could promote the proliferation of lymphocytes and stimulate cell-mediated immunity of SPF chickens. Furthermore, analysis of electron spin resonance (ESR) spectroscopy and physicochemical properties of PAW suggested reactive oxygen and nitrogen species (RONS) played an essential role in the virus inactivation. Therefore, this study indicated that NDV treated by PAW in an appropriate ratio retained immunogenicity on the premise of virus inactivation. PAW as a promising strategy could be used to prepare inactivated vaccine for Newcastle disease.