Abstract: Plant viruses have a great adverse impact on vegetable production. Non-pharmaceutical measures are mainly adopted for vegetable virus prevention and control at present. However, the efficacy of broad-spectrum antiviral agents is limited. The new viral inhibitors are expected to be developed based on novel drug targets. G-quadruplex (G4s) is a non-canonical nucleic acid structure. The replication, transcription, and translation of genes can be regulated by the formation or unfolding of G-quadruplexes in the genome, which affects virus proliferation. Small molecules mediating the stability of G-quadruplex structures exhibit antiviral activity, making G-quadruplexes to be potential antiviral targets. As a widely distributed plant virus, tomato bushy stunt virus (TBSV) is considered as a model virus to study the interaction between the virus and host. The molecular mechanisms of its genome replication, transcription, and translation have been widely studied. Revealing the structure and function of G-quadruplex in TBSV is expected to lay a foundation for the research of G-quadruplex in plant virus genes. In this work, two conserved potential G-quadruplex sequences (putative G-quadruplex sequences, PQS) TBSV-PQS2 and TBSV-PQS4 were identified in TBSV genes through bioinformatics analysis. The G4 ligands interacting with TBSV-PQS2 and TBSV-PQS4 were screened out by UV fluorescence spectrum and circular dichroism (CD) spectrum. Invasive cloning experiments in tobacco found that G-quadruplex ligand N-methyl mesophorphyrin IX (NMM) inhibits TBSV virus proliferation. This work shows that the development of ecological pesticides against plant viruses by targeting the G-quadruplex in plant viruses is expected to become a new strategy for controlling the damage of plant viruses.