Prussian Blue (PB) based glucose biosensor was prepared by immobilizing glucose oxidase (GOD) in layer-by-layer (LBL) films with chitosan (Chi) and multi-walled carbon nanotubes (MWNTs). With the increasing of Chi/MWNTs/GOD layers, the response current to glucose was changed regularly and reached a maximum value when the number of layer was six. At the optimized condition, the biosensor exhibits excellent response performance to glucose with a linear range from I to 7 mM and a low detection limit of 0.05 mM. The biosensor also shows a high sensitivity of 8.017 mu A mM(-1) cm(-2), Which is attributed to the biocompatible nature of the LBL films. Furthermore, the biosensor shows rapid response, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species such as ascorbic acid and acetaminophen. (c) 2007 Elsevier Ltd. All rights reserved.