Both human telomere and proto-oncogene c-MYC can form G-quadruplex (G4) with various conformations. Porphyrin derivative (TMPyP4) could stabilize G4, and thus is considered as a potential drug for anticancer therapeutics. In this paper, the translocation behaviors of three typical G4s (telomere basket, telomere hybrid-1 and c-MYC Pu22 parallel) and their interaction with TMPyP4 were investigated with a single protein nanopore sensing interface with the same main electrolyte of 0.5 M tetramethylammonium chloride. As observed by the statistics of the dwell time of the current pulses, in the presence of K+, the parallel G4 is more stable than the hybrid-1 G4, while the basket G4 in the presence of Na+ exhibited shortest duration. The dwell time of all of the G4s increased as the result of interaction with TMPyP4, indicating an obvious stabilizing effect. This study demonstrated that the single nanopore sensing interface not only reveal the stability of various G4 conformations at a single-molecule level, but also provide the interaction information of a ligand, which could be useful in the drug design. Display omitted •The translocation behaviors of three G4s G4 were examined, and compared for the first time with nanopore sensing interface.•Interaction of porphyrin derivative TMPyP4 with G4s was investigated and compared with the proposed method.•This work provides a new method for obtaining knowledge about the stability of G4 itself and its interaction with ligand.