This work focuses on the influence of the nature of the lipidic matrix and of the hydrophobic or hydrophilic lipidic environment on choline oxidase activity retained in Langmuir-Blodgett (LB) films prepared with behenic acid or with mixed dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA). The enzyme activity was detected through an electrochemical sensor whose platinum electrode was coated with the protein-lipid LB films. It was shown that the hydrophilic environment provided by the phospholipidic LB films was directly responsible for an inactivation of choline oxidase likely due to a competition with the choline moiety of DPPC. The environment provided by the carboxylic groups of the fatty acid multilayers allowed retention of the higest activity. The absence of stability of choline oxidase activity in behenic acid LB films was explained by repulsive electrostatic interactions between the protein and the lipidic molecules, which appeared during the immersion of the LB films in the alkaline buffer required to detect the enzyme activity in optimal conditions. These unfavorable interactions, previously shown to be responsible for a lipidic structural reorganization of choline oxidase-behenic acid LB films, seem to be here responsible for a likely unfolding, yielding protein denaturation. Because it controls the electrostatic interactions between the protein and the lipidic molecules, which may be very unfavorable even in the optimal conditions of the enzyme activity, pH acts as a key parameter of the expression of the enzyme activity in the LB films.