Here we report the use of DNA nanostructures as platforms to monitor the inherent conformational changes of aptamers upon analyte binding, with single-molecule resolution and real-time capability. An aptasensor designed to sense cortisol was found to suffer from instability in solution, but this was reconciled via a rational design of a single-molecule sensing platform. In this regard, DNA origami was employed to immobilise individual aptasensors on a glass surface and to ensure adequate interaction with their environment, for single-molecule analysis. The strategy presented here can be applied to any aptamer obtained by the destabilisation of a duplex in a SELEX process, and hence employed in the rational design of single-molecule biosensors.