NADH/NAD+ is pivotal to fundamental biochemistry research and molecular diagnosis, but recognition and detection for them are a big challenge at the single-molecule level. Inspired by the biological system, here, we designed and synthesized a biomimetic NAD+/NADH molecular clamp (MC), octakis-(6-amino-6-deoxy)-γ-cyclomaltooctaose, and harbored in the engineered α-HL(M113R)7 nanopore, forming a novel single-molecule biosensor. The single-molecule measurement possesses high selectivity and a high signal-to-noise ratio, allowing to simultaneously recognize and detect for sensing NADH/NAD+ and their transformations.