The self-assembled DNA nanostructure has been one of the most interesting research areas in the field of nanoscience, and the application of the DNA self-assembled nanostructures in biosensing is still in the early stage. In this work, based on the target-induced autonomous disassembly of the aptamer–DNAzyme supersandwich nanostructures, we demonstrated a highly sensitive strategy for electrochemiluminescent (ECL) detection of ochratoxin A (OTA). The aptamer–DNAzyme supersandwich nanostructures, which exhibited significant ECL quenching effect toward the oxygen/persulfate (O₂/S₂O₈²⁻) system, were self-assembled on the gold electrode surface. The presence of the target OTA and the exonuclease (RecJ_f) resulted in autonomous disassembly of the nanostructures and cyclic reuse of OTA, leading to efficient recovery of the ECL emission and highly sensitive detection of OTA. Our developed method also showed high selectivity against other interference molecules and can be applied for the detection of OTA in real red wine samples, which offers the proposed method opportunities for designing new DNA-based nanostructures for biosensing applications.