A novel sensing strategy for electrochemical aptamer-based sensors is presented. Nucleic acid aptamers are considered alternatives to antibodies. However, some of their intrinsic properties, such as that they can undergo conformational changes during the binding of the target, can be used to design novel sensing strategies. Unlike other electrochemical "signal off" aptamer-based sensors, we report a strategy based on enzymatic inhibition. Our approach shows the feasibility to detect small molecules based on the aptamer conformational change induced by the target that leads to the inhibition of the enzyme used as a label. Additionally, we prove the ability to regenerate the function of the aptasensor by simply applying a short potential pulse. As a proof-of-concept, the widely used aptamer for ochratoxin A (OTA) has been selected as a model. After self-assembling short oligonucleotides onto a gold electrode, complementary to the 3′ end of the aptamer, hybridization of the aptamer takes place. To investigate the mechanism induced by the OTA-binding, surface plasmon resonance assays were performed, which confirmed the conformational switch of the aptamer rather than the aptamer displacement by dehybridization from the DNA-modified sensor surface. The electrochemical sensor can successfully detect OTA in wine at the limits stipulated by the European Commission. Given its sensitivity, rapid and easy detection, and regeneration, it can be envisaged as screening tool for OTA detection. Moreover, this sensing strategy has the potential to be applied to other aptamer-based biochemical assays for the detection of small molecules in the fields of food safety, environmental monitoring, and medical diagnostics.