High-throughput single-molecule techniques are expected to challenge the demand for rapid, simple, and sensitive detection methods in health and environmental fields. Based on a single-DNA-molecule biochip for the parallelization of tethered particle motion analyses by videomicroscopy coupled to image analysis and its smart combination with aptamers, we successfully developed an aptasensor enabling the detection of single target molecules by a sandwich assay. One aptamer is grafted to the nanoparticles tethered to the surface by a long DNA molecule bearing the second aptamer in its middle. The detection and quantification of the target are direct. The recognition of the target by a pair of aptamers leads to a looped configuration of the DNA-particle complex associated with a restricted motion of the particles, which is monitored in real time. An analytical range extending over 3 orders of magnitude of target concentration with a limit of detection in the picomolar range was obtained for thrombin.