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Sleep debt (SD) is one of the important triggers for causing not only physiological and mental illness but also dangerous work. Therefore, achieving an early and objective assessment of SD is of great significance in the precaution against SD-related diseases and unsafe work. Here, an ultrasensitive electrochemical immunosensor was constructed for analysis of SD biomarker amyloid-β (A β). The gold nanoparticles/chitosan-coated polyaniline-functionalized activated carbon (AuNPs/AC@PANI@CS) composites were employed as the sensing platforms. Since PANI and AC can form an effective conductive path, it can effectively enhance the penetration of electrolytes on the electrode surface and the rapid transport of charges and ions, significantly enhancing the electrochemical response signal of the immunosensor. Under the optimized experimental conditions, the fabricated immunosensor had a wide linear range of 1.95 pg mL−1 to 1000.00 pg mL−1, with a low detection limit of 0.014 pg mL−1. This study not only provides an effective method for the accurate and rapid detection of A β , but also offers a novel evaluation strategy for the objective assessment of SD and the study of related pathological mechanisms. AuNPs/AC@PANI@CS signal amplified electrochemical immunosensor for sensitive detection of amyloid-β (Aβ). [Display omitted] • A highly sensitive and selective electrochemical immunosensor for the determination of amyloid-β (A β) was fabricated. • The prepared AC@PANI with a large specific surface area, excellent electrical conductivity and structure can amplify the signal of the immunosensor. • The proposed immunosensor can be used to sensitively and efficiently analyze A β in serum samples. • This method would provide an effective method for the objective assessment of sleep debt. [ABSTRACT FROM AUTHOR]