Amyloid β-peptide (Aβ) is a key predictor for preclinical diagnosis of Alzheimer's disease (AD) and vascular diseases. In this work, we propose a gold nanoparticle (AuNPs)-Aβ-nickel (Ni)-horseradish peroxidase (HRP) based colorimetric assay for the detection of Aβ. The consecutive binding of Aβ to AuNPs and metal ions is designed and examined for Aβ-specific aggregation of AuNPs and the generation of quantitative colorimetric signals. The affinity of Aβ towards various metal ions was studied first, and two metal ions, Cu and Ni, were specifically tested with Metal Ion-Binding Site Prediction (MIB) and High-resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS). Subsequently, the binding of Aβ and AuNPs was examined, and the binding between Aβ-AuNPs and Ni-HRP was finally analyzed by UV-Vis and nano-zetasizer. Based on the characterized dual binding of Aβ, a colorimetric sandwich assay was developed and the analytical performance of the developed assay has been evaluated with standard solutions and human serum samples. Good linearity within a range from 0 nM to 10 nM was found. The detection limits of 0.22 nM in the standard sample and 0.23 nM in the human serum sample have been demonstrated. The newly developed colorimetric sandwich assay is a short, simple, antibody-free assay and achieves high sensitivity with only 100 μL Aβ samples. The assay has immense potential for the detection of Aβ in biological or biomedical diagnosis.