Two ruthenium(II) complexes [Ru(phen)2(tip)](ClO4)2 (1) and [Ru(bpy)2(tip)](ClO4)2 (2; phen=1,10-phenanthroline, bpy=2,2’-bipyridine, tip=2-thiophenimidazo[4,5-f][1,10]phenanthroline) were synthesized and characterized by elemental analysis, 1H NMR spectroscopy, and electrospray ionization-mass spectrometry to explore the role of metal complexes as novel telomeric quadruplex stabilizers. The different quadruplex binding properties of these compounds were evaluated by absorption and emission analyses, circular dichroism spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, NMR spectroscopy, and molecular modeling. The results show that both complexes can well induce and stabilize different G-quadruplex structures using a 1:1 [quadruplex]/[complex] binding mode ratio. Complex 1 exhibits higher interaction ability at 1.43×106 M−1 binding affinity and superior G-quadruplex selectivity over duplex DNA through multiple interaction (mainly intercalating) with the G-quadruplex at the 3’-terminal face. Furthermore, polymerase chain reaction (PCR)-stop assay, electrophoretic mobility shift assay, telomerase repeat amplification protocol, and MTT assay demonstrate that complex 1 not only can stabilize dimer forms of the G-quadruplex at low concentrations but also exhibit better inhibitory activity for telomerase and cancer cells. The results suggest that complex 1 may be a potential telomerase inhibitor for cancer chemotherapy.