Sustainable agriculture relies on techniques and technology that combine efficiency with the smallest possible ecological and environmental impact. RNA interference (RNAi), a post-transcriptional eukaryotic pathway in eukaryotes can be co-opted for the control of plant pathogens in the topical application system. RNAi is the biological mechanism of mRNA degradation triggered by endogenous or exogenous double-stranded small interfering RNAs (dsRNA/siRNA) and regulates the target gene expression. Topically applied double-stranded RNA (dsRNA) molecules on plants imitate the viral RNA intermediate which is involved in plant virus replication and thereby triggers post-transcriptional gene silencing via., RNAi pathway and reducing viral replication. In this study, a non-transgenic-based RNAi approach was adopted to induce resistance against PVY in potato crop. dsRNA was successfully produced through in vitro transcription and hybridization. To test the effect of dsRNA molecules, in vitro synthesized dsRNA were applied on potato leaves along with PVY. dsRNA-treated plants showed higher resistance to viral disease and exhibited normal growth compared with PVY-treated plants. Therefore, the topical application of dsRNA molecules from the PVY CP gene could serve as a promising method for suppressing PVY infection and ultimately enhancing potato yield.