Langmuir-Blodgett (LB) films with the structure, monolayer of surfactant 1/adsorbed protein layer/monolayer of surfactant 2, were deposited and studied by ellipsometry, IR spectroscopy and atomic force microscopy (AFM) techniques. To avoid protein denaturation, a new method of deposition was applied in which protein molecules avoided contact with the air-water interface and were adsorbed onto the hydrophilic surface of the deposited monolayer in optimal conditions. Five surfactants, cytochrome c, alcohol dehydrogenase and glutathione S-transferase (GST) were used in these structures. The results of measurements on the enzymatic activity of GST show that soluble proteins can be included in LB films using the proposed method without considerable denaturation.