Abstract: Plasma-activated water (PAW) is a new non-thermal sterilization technology, yet its application in pre-prepared duck products and its effect on the quality characteristics of duck fillets are rarely reported. In this study, PAW was prepared using plasma jets with varying discharge durations (0~6 min). The antibacterial activity of PAW was evaluated by analyzing its active substances, and examining the reactive oxygen species (ROS) levels, Fourier transform infrared (FTIR) spectroscopy, and microstructure changes of Escherichia coli (E. coli) treated with PAW. Then, duck fillets were employed as the research object. The effects of PAW on the storage quality of duck fillets were analyzed by measuring the total viable count of E. coli, malondialdehyde (MDA) content and total volatile base nitrogen (TVB-N) content. The results showed that the main active substances (hydrogen peroxide, nitrite and nitrate) in PAW were up to 108.00, 1300.23 and 2396.04 μmol/L at 6 min treatment, respectively. The E. coli in PAW-4 min and PAW-6 min groups were completely inactived (the viable count of E. coli reduced from 6.87 lg CFU/mL to non-detected). Furthermore, PAW treatment elevated intracellular ROS levels, disrupted internal molecular structures, and caused morphological damage to E. coli. During storage, the PAW-6 min group demonstrated the best antibacterial effect, reducing the total viable count of E. coli by 0.51 lg CFU/g compared to the sterile water group on the 6 days of storage period (P<0.05). Additionally, the PAW-treated duck fillets exhibited slower increases in MDA and TVB-N levels after 6 days compared to the control groups, reaching 0.33~0.38 mg/kg and 23.4~25.1 mg/100 g, respectively. The results indicated that PAW improved lipid and protein stability. In conclusion, PAW can effectively extend the shelf life of duck fillets and present a promising novel sterilization technology for the production of pre-prepared duck products.