A glucose biosensor based on layer-by-layer (LBL) self-assembling of chitosan and glucose oxidase (GOD) on a Prussian blue film was developed. First, Prussian blue was deposited on a cleaned gold electrode then chitosan and GOD were assembled alternately to construct a multilayer film. The resulting amperometric glucose biosensor exhibited a fast response time (within 10 s) and a linear calibration range from 6 mu M to 1.6 mM with a detection limit of 3.1 mu M glucose (s/n = 3). With the low operating potential, the biosensor showed little interference to the possible interferents, including ascorbic acid, acetaminophen and uric acid, indicating an excellent selectivity.