AbstractThe synthesis and characterization of a 26‐base DNA hairpin containing both a redox‐active reporter (ferrocene) and terminal thiol functionality for electrochemical gene detection is described. This electrochemical DNA sensor exploits electron‐transfer dynamics that alter as a consequence of a large structural rearrangement (hairpin‐to‐duplex) induced by hybridization of the target DNA sequence. Melting temperature and circular dichroism studies confirm that the 26‐mer DNA forms a hairpin structure in the absence of target DNA. The loop region of the DNA hairpin is shown to form a stable duplex in the presence of complementary single‐stranded DNA. Atomic force microscopy and ellipsometry experiments of immobilized self‐assembled DNA monolayers suggest that hybridization with complementary DNA affords a conformational change that alters the electrochemical response.