<p>Self-powered electrochemical biosensors have emerged as a cutting-edge direction in disease biomarker detection due to their miniaturization potential and external power-free characteristics. Enzymatic biofuel cell-based self-powered biosensors (EBFC-SPB), a green energy conversion device that utilizes biological enzymes as catalysts to directly convert bioenergy into electricity, have become a new research hotspot owing to its operational simplicity, biocompatibility, and efficient performance in both plants and animals. However, challenges such as suboptimal catalytic efficiency, limited open-circuit voltage, and low power output have hindered its applications in human health monitoring. In recent years, researchers have developed various sensitization-enhancement strategies (including nanomaterial sensitization, nucleic acid signal amplification, enzyme catalytic enhancement, energy storage/conversion optimization, and synergistic multi-strategy approaches) to improve EBFC-SPB’s detection capabilities and expand its practical applications in biofluids (such as blood, sweat, saliva, tears, interstitial fluid, and urine), thereby making significant contributions to real-time tracking and accurate detection of disease biomarkers. This review summarizes the working principles of EBFCs, analyzes the design rationale of multi-strategy synergistic sensitization in EBFC-SPB, examines its applications in human disease biomarker detection, and prospects its future research directions and potential applications.</p> Graphical Abstract <p></p>