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Amyloid-β oligomers (AβO) have become representative biomarkers for early diagnosis of Alzheimer's disease. Here, we report on an aptasensor based on stem-loop probes for sensitive and specific detection of AβO by an amperometric transducer principle using alternating current voltammetry (ACV). Stem-loop probes with redox-active moieties are immobilized on a gold substrate as a receptor element. The signal transduction mechanism relies on redox ferrocene (Fc) reporting via charge transfer on a molecular recognition event involving a conformational change of the molecular beacon. The stem-loop structures were optimized by considering the aptamers' stem length, spacer, and different ferrocene terminals. In addition, the sensor assembly and signal recording including aptamer concentration and ACV frequency dependence are discussed. Using the optimized stem-loop probe (B-3′ Fc), the aptasensor showed a decrease of the Fc peak current induced by AβO binding within the broad concentration range spanning 6 orders of magnitude. Furthermore, the detection limit of the sensor can be further decreased by optimizing the ACV frequency, however at the cost of a narrowed detection range. In this work, a label-free electrochemical aptasensor is demonstrated, which facilitates the quantification of the concentration of AβO with high selectivity and subpicomolar sensitivity, which may be conducive to improving the diagnosis and pharmacology studies of Alzheimer's disease.