Enzyme multilayer films were prepared on the surface of a glassy carbon electrode by a layer-by-layer deposition of concanavalin A (Con A) and horseradish peroxidase (HRP) or glucose oxidase (GOx) alternately. The enzyme thin films were formed through biological affinity between Con A and sugar chains intrinsically located on the surface of the enzymes. Both HRP and GOx retained their catalytic activity in the multilayer film and the enzyme-modified electrodes served as enzyme biosensors. For the glucose sensors prepared using HRP-GOx bienzyme multilayer films, the loading and geometry of the enzymes in the multilayer film were optimized by varying the number of enzyme layers and the sequence of immobilization in the multilayer film. The magnitude of response of the glucose sensors increased with the increasing number of Cox layers, while the bilayers of HRP sufficed for attaining maximum response. It was found that the electrode surface should be first coated with HRP layers and then the GOx layer should be immobilized as an outer layer to obtain higher performance sensors. The results were explained reasonably based on the relative activity of the enzymes and the reaction scheme.