Amyloid β-peptide (Aβ) in its oligomeric form is often considered as the most toxic species in Alzheimer’s disease (AD), and thus Aβ oligomer is a potentially promising candidate biomarker for AD diagnosis. The development of a sensitive and reliable method for monitoring the Aβ oligomer levels in body fluids is an urgent requirement in order to predict the severity and progression at early or preclinical stages of AD. Here, we show a proof of concept for a sensitive and specific detection of Aβ oligomers by an antibody-aptamer sandwich assay. The antibodies of Aβ oligomers and a nanocomposite of gold nanoparticles with aptamer and thionine (aptamer-Au-Th) were used as the recognition element and the detection probe for specifically binding to Aβ oligomers, respectively. The electrochemical signal of Th reduction could provide measurable electrochemical signals, and a low limit of detection (100 pM) was achieved due to the signal amplification by high loading of Th on the gold nanoparticles. The feasibility of the assay was verified by test of Aβ oligomers in artificial cerebrospinal fluid. The proposed strategy presents valuable information related to early diagnosis of AD process.