Abstract: Previous studies have shown that NbNAC062 inhibits early PVY infection. In this study, NbNAC062 knockout mutants and overexpression plants were used to further clarify the antiviral function of NbNAC062. The NbNAC062 plasmid was coated with fluorescein isothiocyanate (FITC)-labeled chitosan quaternary ammonium salt (HACC) to prepare HACC-NbNAC062 nanomedicine. Laser confocal microscopy was used to trace the nanomedicine and its characteristics were analyzed by transmission electron microscopy and laser particle analyzer. The viral content was determined GFP fluorescence difference, qRT-PCR and Western blot to explore the effects of nanomedicine on PVY infection. The results showed that the viral GFP fluorescence in the knockout group was enhanced, while the viral fluorescence in the overexpression group was weakened. The PVY CP content was consistent with the above results. The diameter of HACC-NbNAC062 nanomedicine was 18~32 nm; the Zeta potential was +41.8 mV. FITC-HACC (green fluorescence) and RFP-NbNAC062 (red fluorescence) were observed in the plant cells at 48 h after infiltrated with the HACC-NbNAC062 nanomedicine. Comparing with the control group, the PVY CP mRNA levels in the NbNAC062-HACC group were down-regulated by 17.41%, 47.81%, and 13.03% respectively 5, 7, and 9 days after PVY-GFP inoculation. The protein level was significantly lower than that of the control group at 7 days after viral inoculation, and the GFP fluorescence intensity was significantly darker than that of the control group. The above research results indicated that the HACC-NbNAC062 nanomedicine successfully delivered NbNAC062 and exerted its inhibitory effect on the initial infection of PVY.