Immobilization and electrochemical characterization of specially designed functional DNA-aptamer constructs are of great importance for the development of versatile biosensors (not limited to gene analysis) and the investigation of molecular interactions between DNA and other molecules. We have constructed a "DNA conformational switch" by incorporating the antiadenosine aptamer sequence in the middle of an otherwise cDNA double helix, as its structural change responds to the presence of small molecule ligands (e.g., adenosine). In particular, methylene blue (MB) was used as a model system to probe the rather complex interaction modes between small redox molecules and the dsDNA-aptamer construct. Besides intercalating with the double-stranded DNA stem, MB can stack with a single guanine base in the relatively unstructured aptamer domain or electrostatically bind to the DNA backbone. The decreased surface density of MB after adenosine binding indicated that the ligand-gated structural change of the dsDNA-aptamer construct can eliminate MB molecules that were originally bound to the aptamer domain but not those in the complementary stem.