Small molecules targeting G-quadruplexes (G4s) in viruses could inhibit viral proliferation. The 1a protein of cucumber mosaic virus (CMV) act as RNA-dependent RNA polymerase (RdRp) that plays a crucial role in regulating the replication of CMV. In this study, four putative G4 sequences (CMV PQS1–PQS4) in the genetic coding region of CMV 1a were identified, and three of them (PQS2, PQS3, and PQS4) were confirmed to fold into G4 structures. The G4-ligand, RHPS4, could bind to CMV PQS2 and PQS4 with a strong binding affinity and preferred to interact with the 3′ terminal G-quartet surfaces of CMV PQS2, and 5′ terminal of CMV PQS4. RHPS4 was also found to stabilize the CMV PQS2 and PQS4 G4s. Further studies revealed that RHPS4 exhibited an excellent anti-CMV activity. This study suggested that CMV PQS2 and PQS4 could be considered potential targets for screening viral inhibitors.