<p>Current methods for detecting biotoxins are hindered by complex sample preparation and dependence on sophisticated instrumentation. Consequently, there is an urgent need for innovative solutions that facilitate efficient and portable detection capabilities. Herein, a self-powered biosensing platform based on enzyme-based biofuel cells (EBFCs) is reported for the first time for ultrasensitive detection of aflatoxin B1 (AFB1), utilizing gold-embedded hierarchical porous carbon (Au@HPC) and enzymatic signal amplification. Au@HPC was prepared to function as an electrode material of EBFCs for constructing superior EBFCs, representing a novel application of this composite in a self-powered sensing platform. An aptamer-based selective recognition strategy coupled with enzymatic signal amplification was further implemented to monitor the bioelectrocatalytic response toward AFB1. Synergy of enzymatic signal amplification and high-performance carrier material (Au@HPC) amplified electrical signals, markedly improving the sensitivity of the detection platform. As a result, AFB1 could be reliably identified over a wide concentration range of 0.01–10⁴&#xa0;pg/mL, with a detection limit reaching the femtogram level (1.52&#xa0;fg/mL, S/N = 3). This study presents a highly accurate, sensitive, and portable detection method suitable for rapid analysis in real food samples.</p> Graphical abstract <p></p>