Biological nanopores are emerging as a valuable tool for detection and characterization of single-molecule processes. In particular, the ability of observing individual molecules provides a distinctive advantage compared to ensemble techniques, where rare intermediates would be lost in ensemble averaging. The basic concept of nanopore analysis is to observe, under an applied potential, the disruption of the flow of ions through the pore caused by the capture of an analyte within the lumen of the pore. Of particular interest are folded DNA structures or aptamers that are selected to bind molecular targets (e.g. proteins) with high affinity, and that are used as diagnostic tools or therapeutic agents. One of the most studied aptamers is the thrombin binding aptamer (TBA) that binds to thrombin, a key enzyme in the blood coagulation cascade, which malfunction can lead to severe blood clotting abnormalities. In this work we introduced a simple and cost-effective technique to investigate the interaction of thrombin binding aptamers with thrombin, based on the ClyA nanopore. We showed that the affinity constant and apparent lifetime for the interaction of TBA with thrombin can be rapidly measured, while the single-molecule nature of nanopore recordings revealed that TBA binds to thrombin in two orientations, which have distinctive binding constants. The use of TBA mutants allowed us to understand the exact binding mechanism of TBA to thrombin in detail. Therefore this work suggests that nanopore recordings can offer a new tool to study DNA:protein interactions with high bandwidth at the single-molecule level.