The enzyme glutamate dehydrogenase (GlDH) was adsorbed onto hydrophilic or hydrophobic surfaces of behenic acid Langmuir-Blodgett (LB) films. FTIR spectroscopy shows that the GlDH molecules can be retained onto both types of surfaces without modifying the LB film structure; the enzyme adsorption is only responsible for a superficial hydration of the multilayers. Epi-fluorescence microscopy shows that the enzyme remains active. The enzyme retention was investigated in the conditions of enzyme activity detection by immersion of the protein-lipid LB films in the suitable alkaline buffer. This study showed that the interactions between the enzyme and either the head or the tail groups of behenic acid were strong enough to avoid enzyme desorption during overnight immersion in the buffer. Furthermore, the retained enzyme preserves the structure of the behenic acid multilayers during their immersion in the alkaline buffer; transformation into behenate multilayers which normally occurs if the enzyme is not present. This protecting role of the GlDH molecules against the modification of the behenic acid multilayer structure, probably contributes to a slowing down of the hydration of the underlaying layers by the buffer.