Single-molecule localization allows determining the underlying biological and biochemical processes and promotes the development of super-resolution imaging techniques. Here, we present an optical technique of tracking the motion of a single nanoparticle linked to a substrate via a biomolecule tether to reveal the localization of single biomolecules and the transient states of single nanoparticle switching between specific binding pairs. The affinities, steric hindrance, and conformational variation of a single-molecule binding pair uncover the dynamic details and intrinsic mechanism of binding processes with high specificity and accuracy (a few nanometers). The application of tracking motions of single soft liposomes on different modified surfaces was further demonstrated, which revealed the characteristic behavior related to surface chemistry. Our results show that the trajectory of nanoscale liposomes loaded with small-drug molecules is linked to the compositional inhomogeneity, which provides a route for thorough comprehension of the fundamental biotechnological process.