BACKGROUND: The development of highly efficient and environmentally friendly plant virus inhibitors with novel structures mostly depends on the discovery of new targets. G-quadruplexes are non-classical high-level structures that are formed by guanine-rich nucleic acid sequences, which have significant biological functions. Small molecules that target the G-quadruplexes in the genomes of animal viruses have been shown to inhibit viral proliferation. However, the structure and function of the G-quadruplex in the plant virus genome have rarely been reported. Investigating the function of plant virus G-quadruplexes facilitates the discovery of new targets for screening viral inhibitors. RESULTS: In this study, we found that the 3'-end untranslated regions (3'UTRs) of cucumber mosaic virus (CMV) 1a, 2b, and CP genes all contain a putative G-quadruplex sequence (PQS) composed of the same base sequence, which was identified to fold into a G-quadruplex structure (CMV-3'UTR-PQS). In comparison to other G-quadruplex ligands, BRACO-19 exhibited the strongest binding affinity for the CMV-3'UTR-PQS. Further studies revealed BRACO-19 increased the ability of CMV-3'UTR-PQS to inhibit the expression of gene and exhibited an excellent anti-CMV activity. Moreover, BRACO-19 was found to induce the formation of intermolecular G-quadruplex structure from intramolecular G-quadruplex in CMV-3'UTR-PQS, and shifted the balance of the conformational ensemble of CMV-3'UTR-PQS in the direction of the compact conformation. CONCLUSION: This study suggested that CMV-3'UTR-PQS can be used as a target for screening viral inhibitors and provided new strategies for CMV prevention and control. © 2025 Society of Chemical Industry.