Fresh walnuts exhibit unique flavor and multiple biological activities, but they are highly perishable due to the lack of practical and cost-effective postharvest preservation technique. In this paper, the quality of fresh walnut kernels treated with deionized water, water containing ClO2, and plasma-activated water (PAW) for 15 min were monitored during 4 °C storage. Results showed that 100-s PAW treatment reduced the total viable count by 1.15 log CFU/g immediately after treatment. The bactericidal and bacteriostatic effects of PAW was superior to 4 mg/L ClO2. PAW-treated samples retained their flavor during 12d storage at 4 °C, while other samples generated odor at 6d. Moreover, PAW treatment could prevent nutritional loss, browning, and rancidity. The increases of peroxidase, polyphenol oxidase, and lipase activities were inhibited after PAW treatment. Hence, PAW treatment can be applied for the preservation of fresh walnut kernels due to its effect on microbial inhibition and quality maintenance. Industry relevanceNon-thermal plasma is an emerging non-thermal food processing technology due to its advantages of non-destructive, pollution-free, high efficiency and short treatment periods. In this study, plasma-activated water (PAW) was applied in the preservation of fresh walnut kernels, and inhibited flavor changes, nutritional loss, microbial growth, browning, and oxidation reaction. When approaching food preservation technologies, especially for heat-sensitive materials, PAW treatment as a novel technique appears to have far-reaching implications for increasing markets by antimicrobial properties, and avoiding the negative impacts on quality attributes.