The molecular engineering capability of the layer-by-layer (LbL) method for fabricating thin films has been exploited in order to immobilize glucose oxidase (GOD) in films with alternating layers of chitosan. Chitosan was proven a good scaffolding material, as GOD molecules preserved their catalytic activity towards glucose oxidation. Using electrochemical measurements, we showed that chitosan/GOD LbL films can be used to detect glucose with a limit of detection of 0.2 mmol l(-1) and an activity of 40.5 mu A mmol(-1) L mu g(-1), which is highly sensitive when compared to other sensors in previous reports in the literature. The highest sensitivity of the LbL film was achieved when only the top layer contained GOD, thus indicating that GOD in inner layers did not contribute to glucose oxidation, probably because it hampers analyte diffusion and electron transport through the deposited layers. This may be explained by the dense packing of GOD molecules in the LbL films with chitosan, as inferred from estimates of the amount of GOD adsorbed per layer using a quartz crystal microbalance. (c) 2006 Elsevier Inc. All rights reserved.