This study deals with the structural modifications of enzyme-lipid Langmuir-Blodgett films under conditions required for enzyme activity detection. As a model, choline oxidase was associated with behenic acid LB films either through a direct adsorption onto the head groups of the lipidic LB films or through an adsorption followed by the transfer of a behenic acid layer onto the protein molecules, which sandwiched the enzyme between the polar groups of two lipidic layers. The structure, homogeneity, and stability of the protein-lipid LB stacking has been studied using FTIR spectroscopy and Nomarski differential interference contrast microscopy before and after immersion of the proteo-lipidic multilayers in the buffer used for the enzymatic activity detection. In the absence of enzyme, a pH variation induces a structural reorganization of the Lipid stacking with formation of lipidic vesicles, most probably in reversed phase. The association of choline oxidase with the fatty acid LB films acts as an accelerating factor of this lipidic reorganization. These results can be explained by the effects of both hydration of the multilayers and the establishment of repulsive interactions either between protein and lipid molecules or between lipid molecules themselves.