A multilayered glucose biosensor via sequential deposition of Prussian blue (PB) nanoclusters and enzyme-immobilized poly(toluidine blue) films was constructed on a bare Au electrode using electrochemical methods. The whole configuration of the present biosensor can be considered as an integration of several independent hydrogen peroxide sensing elements. In each sensing element, the poly(toluidine blue) film functioned as both the supporting matrix for the glucose oxidase immobilization and the inhibitor for the diffusion of interferences, such as ascorbic acid and uric acid. Meanwhile, the deposited Prussian blue nanocluster layers acts as a catalyst for the electrochemical reduction of hydrogen peroxide formed from enzymatic reaction. Performance of the whole multilayer configuration can be tailored by artificially arranging the sensing elements assembled on the electrode. Under optimal conditions, the biosensors exhibit a linear relationship in the range of 1 x 10(-4) to 1 x 10(-2) mol/L with the detection limit down to 10(-5) mol/L. A rapid response for glucose could be achieved in less than 3 s. For 1 mM glucose, 0.5 mM acetaminophen, 0.2 mM uric acid, and 0.1 mM ascorbic acid have no obvious interferences (<5%) for glucose detection at an optimized detection potential. The present multilayered glucose biosensor with a high selectivity and sensitivity is promising for practical applications.