The alpha-hemolysin (alpha HL) protein pore has many applications in biotechnology. This article describes a single-molecule manipulation system that utilizes the nanocavity enclosed by this pore to noncovalently encapsulate a guest molecule. The guest is the thrombin-binding aptamer (TBA) that folds into the G-quadruplex in the presence of cations. Trapping the G-quadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded DNA in the pore. Through detection with Tag-TBA, we localized the G-quadruplex near the entry of the beta-barrel inside the nanocavity, where the molecule vibrates and rotates to different orientations. This guest-nanocavity supramolecular system has potential for helping to understand single-molecule folding and unfolding kinetics.