Amyloid beta-protein (AβAβ) is a main hallmark of Alzheimer's disease (AD), and a low amount of Aβ protein accumulation appears to be a potential marker for AD. Here, an electrochemical DNA biosensor based on polyamide/polyaniline carbon nanotubes (PA/PANI-CNTs) is developed with the aim of diagnosing AD early using a simple, low-cost, and accessible method to rapidly detect Aβ42 in human blood. Electrospun PA nanofibers served as the skeleton for the successive in situ deposition of PANI and CNTs, which contribute both high conductivity and abundant binding sites for the Aβ42 aptamers. After the aptamers are immobilized, this aptasensor exhibits precise and specific detection of Aβ42 in human blood within only 4min with an extremely fast response rate, lower detection limit, and excellent linear detection range. These findings make a significant contribution to advancing the development of serum-based detection techniques for Aβ42, thereby paving the way for improved diagnostic capabilities in the field of AD.