This study explores the use of anaerobic digestion (AD) pretreatment to enhance the performance of lignocellulosic biomass-derived biochar as an electrocatalyst for oxygen reduction reaction (ORR) and microbial fuel cells (MFCs). AD pretreatment effectively degrades the biomass structure, enriches nitrogen content, and creates a porous structure, leading to improved physicochemical properties of the resulting biochar. The biochar obtained after 15 days of AD treatment showed the highest performance, with an onset potential of 0.17 V (vs. saturated calomel electrode) and a maximum power density of 543.2 mW cm⁻² in MFCs. The enhanced performance is attributed to increased graphitization, nitrogen doping, and the formation of active nitrogen sites. The study demonstrates that AD pretreatment is a viable and cost-effective method for producing high-performance biochar-based electrocatalysts, offering a sustainable pathway for valorizing biomass resources. The results highlight the potential of AD as a biological pretreatment method for biomass-based electrocatalysts, providing a unique approach to fabricate high-performance biochar materials through structure optimization and nitrogen-containing active site construction. This method not only improves the electrocatalytic performance but also contributes to the complete valorization of lignocellulosic biomass.This study explores the use of anaerobic digestion (AD) pretreatment to enhance the performance of lignocellulosic biomass-derived biochar as an electrocatalyst for oxygen reduction reaction (ORR) and microbial fuel cells (MFCs). AD pretreatment effectively degrades the biomass structure, enriches nitrogen content, and creates a porous structure, leading to improved physicochemical properties of the resulting biochar. The biochar obtained after 15 days of AD treatment showed the highest performance, with an onset potential of 0.17 V (vs. saturated calomel electrode) and a maximum power density of 543.2 mW cm⁻² in MFCs. The enhanced performance is attributed to increased graphitization, nitrogen doping, and the formation of active nitrogen sites. The study demonstrates that AD pretreatment is a viable and cost-effective method for producing high-performance biochar-based electrocatalysts, offering a sustainable pathway for valorizing biomass resources. The results highlight the potential of AD as a biological pretreatment method for biomass-based electrocatalysts, providing a unique approach to fabricate high-performance biochar materials through structure optimization and nitrogen-containing active site construction. This method not only improves the electrocatalytic performance but also contributes to the complete valorization of lignocellulosic biomass.