Alzheimer’s disease (AD) is connected to aggregation of amyloid-β (Aβ) peptide and formation of insoluble plaques in the brain. Aβ level can be monitored as an AD early diagnosis route. In this study, an irregular shaped microporous gold nanostructure with a typical size of 150 × 250 nm was electrodeposited on a polycrystalline gold surface at 0 mV (vs. AgCl) using sodium alendronate. The nanostructure was then characterized by field-emission scanning electron microscopy. An electrochemical peptide-based biosensor was fabricated by immobilizing an Aβ(1–42)-binding peptide on the gold nanostructure. Binding of Aβ(1–42) by the peptide was followed electrochemically using ferro/ferricyanide as a redox probe. Differential pulse voltammograms in a potential range of 0–500 mV (vs. AgCl) with typical peak potentials at 224 mV are linear in the 3–7000 pg mL−1 Aβ(1–42) concentration range, with a 0.2 pg mL−1 detection limit. The biosensor is free of interferences and was applied to the quantitation of Aβ(1–42) in artificial cerebrospinal fluid and spiked serum samples.