The bioanode serves as a pivotal component in microbial fuel cells (MFCs), and a comprehensive understanding of its electrochemical properties, along with the establishment of precise theoretical models, is crucial for the practical application of MFC technology. To achieve this, experiments were conducted using a continuous-flow reactor equipped with a large-volume electrolyte and an oversized cathode, aiming to eliminate limitations affecting bioanode performance. Under these conditions, duplicate anodes composed of untreated carbon felt (CF) and phosphate-treated carbon felt (CF-P) were examined. CF-P exhibited a 133% higher maximum power density compared to CF. Both charge transfer impedance (Rct) and diffusion impedance (Zw) continuously decreased with increasing anode potential, with the lowest total impedance values (70.9 Ω for CF and 18.2 Ω for CF-P) observed at 5.7 mV and –102 mV vs. Ag/AgCl, respectively—distinct from previously reported optimal potentials in batch systems. Electrode impedance (Rel) exhibited very low capacitance and remained independent of the anode potential, suggesting abiotic electron transfer via conductive appendages. In contrast, an increase in anode potential decreases Rct by enhancing overpotential-driven current generation, promoting the oxidation of redox cofactors, and reorganizing the electron-hopping network to facilitate more efficient intracellular and interfacial electron transfer. Additionally, as the anode potential increases, the effective diffusivity of intracellular electrons or redox cofactors within the biofilm increases, resulting in a decrease in Zw. Notably, for the 80 minutes of impedance measurement times, the anode biofilm characteristic is changed, and it was confirmed by impedance measurement.