G-quadruplex formation at the end of telomeres is one of the effective pathways for inhibiting the growth of cancer cells induced by up-regulation of telomerase. The lack of effective G-quadruplex-binding ligands has strongly limited the discovery of telomerase-targeted cancer drugs. To address these limitations, the development of a high-throughput screening technique is urgent and meaningful for the discovery of potent telomerase inhibitors from the large chemical libraries of candidates. Here, we present a fast and cost-effective method to realize high-throughout screening of G-quadruplex-binding ligands based on G-quadruplex formation induced fluorescence quenching. Carboxyfluorescein-labeled hairpin DNA (F-hpDNA) was designed as a molecular recognition probe. In the presence of G-quadruplex ligands, the conformation of the F-hpDNA switched from hairpin into G-quadruplex, leading to a significant decrease in the fluorescence of F-hpDNA due to photo-induced electron transfer (PIET) between fluorophore and G-quartet. So, ligands were selected by using the G-quadruplex as a natural fluorescence quencher, which means the DNA probe is just single-labeled. A variety of ligands can be simultaneously screened within several minutes. To verify the formation of G-quadruplex, circular dichroism (CD) analysis has been performed to study ligand-induced conformation change. Moreover, the fluorescence microscopy image and MTT cell proliferation assay all demonstrated that the selected ligand has the potential to inhibit the growth of cancer cells. For the first time, a high-throughout method based on PIET was established to quickly, cost-effectively and reliably identify G-quadruplex ligands, which is of great theoretical and practical importance for the discovery of telomerase-targeted anticancer drugs.