The modification of a gas diffusion layer (GDL), a vital component in polymer electrolyte fuel cells, is described here for use in the electrochemical detection of antibody-antigen biosensors. Compared to other substrates (gold foil and graphite), mouse anti-rHBsAg monoclonal antibody immobilized on gold-coated GDL (G-GDL) detected analytes of goat anti-mouse IgG antibody-ALP using a relatively low potential (-0.0021 V vs. Ag/AgCl 3 M NaCl), indicating that undesired by-reactions during electrochemical sensing should be avoided with G-GDL. The dependency of the signal against the concentration of analytes was observed, demonstrating the possibility of quantitative electrochemical biosensors based on G-GDL substrates. When a sandwich method was employed, target antigens of rHBsAg with a concentration as low as 500 ng/mL were clearly measured. The detection limit of rHBsAg was significantly improved to 10 ng/mL when higher concentrations of the 4-aminophenylphosphate monosodium salt (APP) acting on substrates were used for generating a redox-active product. Additionally, it was shown that a BSA blocking layer was essential in improving the detection limit in the G-GDL biosensor.