Structural characterization of aptamer-protein interactions is challenging and limited despite the tremendous applications of aptamers. Here we for the first time report a fluorescence anisotropy (FA) approach for mapping the interaction of an aptamer and its protein target at the single nucleotide level. Nine fluorescently labeled aptamers, each conjugated to a single tetramethylrhodamine at a specified nucleotide in the aptamer, were used to study their interactions with thrombin. Simultaneous monitoring of both fluorescence anisotropy changes and electrophoretic mobility shifts upon binding of the fluorescently modified aptamer to the protein provides unique information on the specific nucleotide site of binding. T25, T20, T7 and the 3'-end were identified as the close contact sites, and T3, C15T, and the 5'-end were identified as the sites distant from the binding. This approach is highly sensitive and does not require cross-linking reactions. Studies of aptamer-protein interactions using this technique are potentially useful for design, evolution, and modification of functional aptamers for a range of bioanalytical, diagnostic, and therapeutic applications.