The present paper describes the formation of glucose oxidase (GOX)-L-alpha-lecithin Langmuir film on air-water interface by spreading GOx solution directly onto subphase covered with layer of lecithin. The optimum experiment conditions were obtained according to the experimental results. Two phase transition processes were observed under surface pressure ranges of 8.0-11.0 mN/m and 15.0-30.0 mN/ m, which represented the movement of GOx molecules under the lecithin layer and the reorientation of GOx molecules in the lecithin layer and/or expulsion of GOx molecules from the lecithin layer, respectively. The forces of GOx molecules that interacted with the lecithin layer were hydrogen bonding and hydrophobic force. An atomic force microscopy image of GOx-lecithin film deposited on An (111) surface in optimal conditions gives evidence of well-ordered GOx molecules in the lecithin layer. As a target of this research, this work provides a new way to prepare biomimetic film and design glucose biosensors in future. (C) 2006 Elsevier B.V. All rights reserved.