Tremendous developments in biosensors have obviously contributed to the rapid and robust detection of many analytes, but incorporation of signal amplification strategies is still necessary to magnify the weak signal output especially when dealing with trace amounts of analytes. Herein, we developed a bioinspired approach to detect the soluble amyloid-β oligomer (AβO), a significant and reliable biomarker of Alzheimer's disease (AD), based on a novel signal amplification strategy using an in-situ self-assembly of peptides. Taking advantage of the specific interaction between the target AβO and the PrP(95–110) which was a segment of cellular prion protein, a cysteine-containing peptide CP4-PrP(95–110) was designed and firstly immobilized onto the surface of a gold electrode via Au-S bond for capturing the AβO. Subsequently, another ferrocene-conjugated peptide C16-GGG-PrP(95–110)-Fc was employed for both the recognition of the captured AβO and the generation of amplified electrochemical signal due to the in-situ peptide self-assembling and the resulting accumulation of a large number of Fc moieties. After a sandwiched reaction, an enhanced detection sensitivity was obtained with a detection limit of 0.6 nM, which was 8-fold lower than un-amplified strategy. Compared to other reported methods for detecting AβO, the approach in this work is simple, fast, highly selective and sensitive. The proposed peptide self-assembly based signal amplification strategy will be adapted for the detection of other disease-related biomarkers in order to provide valuable information related to the early diagnosis of disease.