Ligands that selectively promote the formation of G-quadruplexes in human telomeres have great potential for cancer treatment by inhibiting the telomere extension by telomerase. Thus, understanding the interactions of the G-quadruplex ligands with the telomere sequence at the single-molecule level is of significant importance. Here, human telomere sequence interactions with a small molecule ligand pyridostatin (PDS) were analyzed via α-hemolysin protein nanopore, and a nanopore thermodynamic analytical method was proposed. The prolonged unraveling time of the telomeric DNA G-quadruplex after PDS binding demonstrated the potent stabilization effect of ligand on G-quadruplex structure. The signature two-level electronic blocks generated by K+-PDS-G-quadruplex complexes suggested a two-state unraveling process, including the dissociation of the interquartet cation and the unraveling of the cation-free ligand-bound G-quadruplex. The translocation studies and the analysis of free-energy changes demonstrated a ligand-binding mode in which PDS molecule and K+ were simultaneously bound to one G-quadruplex structure with the coordinated effect on G-quadruplex stabilization. The single-molecular nanopore platform permits the efficient and accurate determination of ligand affinity constants without the requirement for labeling, amplification, or ligand/receptor titration, which provides a general analytical tool for effectively monitoring and quantifying the G-quadruplex/ligand interactions, possessing important implications for the design and screen of anticancer drugs.