A series of fluorescent ligands, which were systematically constructed from thiazole orange scaffold, was investigated for their interactions with G-quadruplex structures and antitumor activity. Among the ligands, compound was identified to exhibit excellent specificity toward telomere G4-DNA over other nucleic acids. The affinity of -Htg24 was almost 5 times higher than that of double-stranded DNA and promoter G4-DNA. Interaction studies showed that may bind to both G-tetrad and the lateral loop near the 5'-end. The intracellular colocalization with BG4 and competition studies with BRACO19 reveal that may interact with G4-structures. Moreover, reduces the telomere length and downregulates hTERC and hTERT mRNA expression in HeLa cells. The cytotoxicity of against cancer cells (IC = 12.7-16.2 μM) was found to be generally higher than noncancer cells (IC = 52.3 μM). The findings may support that the ligand is telomere G4-DNA specific and may provide meaningful insights for anticancer drug design.