Glucose oxidase (GOx) molecules were adsorbed from aqueous solution onto template monolayer of octadecylamine (ODA) or stearic acid (SA) at the air/liquid interface. The effects of the template layers on the GOx adsorption and the characteristics of the mixed template/GOx monolayers are studied. The monolayers at the air/liquid interface were then transferred onto Pt substrates to prepare GOx Langmuir–Blodgett (LB) films for glucose sensing study. The results show that the SA/GOx monolayer has a pressure–area isotherm resembles that of a SA monolayer, indicating that only a small amount of GOx was incorporated in the mixed film. On the contrary, the ODA/GOx monolayers exhibit high expanded and high compressible characteristics, attributed to the high incorporation amount of GOx induced by the electrostatic interaction between ODA and GOx. The glucose sensing experiments demonstrate that the ODA/GOx LB films have much better performance than SA/GOx films in terms of current sensitivity and current responding rate. Furthermore, an ODA/GOx film prepared after approaching the second equilibrium stage of GOx adsorption (8h adsorption) shows a better performance than that prepared at the first equilibrium stage. Good linear relationship between response current and glucose concentration was obtained for the GOx-LB films between 0.1 and 5mM.