The strong avidin–biotin affinity is used to stack up successive monomolecular layers of horseradish peroxidase on carbon electrodes. After a biotinylated immunoglobulin is adsorbed on the electrode surface, alternate deposition of neutravidin and biotinylated HRP allows the assemblage of up to 16 successive active HRP layers. The film build-up is followed by cyclic voltammetry using an osmium complex as soluble mediator and H₂O₂ as substrate. The variation of the resulting catalytic responses with H₂O₂ concentration exhibit characteristics qualitatively consistent with the catalysis-inhibition reaction scheme previously established for monomolecular layers. In most cases the catalytic activity increases steadily with the number of monomolecular layers, leading to a significant increase of the analytical sensitivity of the derivatized electrode.