Negatively charged myoglobin (at pH values above its isoelectric point) was immobilized with the positively charged polyion polyethyleneimine (PEI) on pyrolytic graphite electrodes. A modified form of the common layer-by-layer technique was proposed, which consisted in forming non-ordered cast polyion films. The modified Mb electrodes obtained by both techniques were compared by cyclic voltammetry. The cast polyion technique gave less reproducible results but enable the immobilization of higher percentages of the total quantity of protein required to prepare the electrodes. The electrochemical properties of the negatively charged myoglobin were determined, and its capability to catalyze the electrochemical reduction of oxygen and the dechlorination of trichloroacetic acid was demonstrated. The mechanisms are discussed, and the modified pathway that has been proposed to take into account the influence of pH in the Mb-catalyzed reduction of oxygen was confirmed here.