This study investigates the use of anaerobic digestion (AD) as a pretreatment method to enhance the performance of biomass-based carbon electrocatalysts. The AD process successfully depolymerized the carbohydrates and enriched nitrogen in *Hybrid Pennisetum* (HP), leading to the formation of porous biochar with improved physicochemical properties. The biochar exhibited higher specific surface areas, nitrogen content, and graphitization degree compared to those obtained from pyrolysis of raw biomass. These improvements were positively correlated with the AD time and enhanced the performance in oxygen reduction reaction (ORR) and microbial fuel cell (MFC) applications. Among the investigated samples, the biochar pretreated with AD for 15 days showed the best performance, with an onset potential of 0.17 V and a maximal power density of 543.2 mW cm\(^{-2}\) in MFCs. The study suggests that AD can be a new biological pretreatment method for preparing high-performance biomass-based electrocatalysts, providing a cost-effective approach to fabricate metal-free catalysts for ORR.This study investigates the use of anaerobic digestion (AD) as a pretreatment method to enhance the performance of biomass-based carbon electrocatalysts. The AD process successfully depolymerized the carbohydrates and enriched nitrogen in *Hybrid Pennisetum* (HP), leading to the formation of porous biochar with improved physicochemical properties. The biochar exhibited higher specific surface areas, nitrogen content, and graphitization degree compared to those obtained from pyrolysis of raw biomass. These improvements were positively correlated with the AD time and enhanced the performance in oxygen reduction reaction (ORR) and microbial fuel cell (MFC) applications. Among the investigated samples, the biochar pretreated with AD for 15 days showed the best performance, with an onset potential of 0.17 V and a maximal power density of 543.2 mW cm\(^{-2}\) in MFCs. The study suggests that AD can be a new biological pretreatment method for preparing high-performance biomass-based electrocatalysts, providing a cost-effective approach to fabricate metal-free catalysts for ORR.