Prussian blue has been formed by cyclic voltammetry onto the basal pyrolytic graphite surface to prepare a chemically modified electrode which provides excellent electrocatalysis for both oxidation and reduction of hydrogen peroxide. It is found for the first time that glucose oxidase or D-amino oxidase can be incorporated into a Prussian blue film during its electrochemical growth process. Two amperometric biosensors were fabricated by electrochemical codeposition, and the resulting sensors were protected by coverage with a thin film of Nafion. The influence of various experimental conditions was examined for optimum analytical performance. The glucose sensor responds rapidly to substrates with a detection limit of 2 × 10−6 M and a linear concentration range of 0.01–3 mM. There was no interference from 2 mM ascorbic acid or uric acid. Another (D-amino acid) sensor gave a detection limit of 3 × 10−5 M D-alanine, injected with a linear concentration range of 7.0 × 10−5-1.4 × 10−2 M. Glucose and D-amino acid sensors remain relatively stable for 20 and 15 days, respectively. There is no obvious interference from anion electroactive species due to a low operating potential and excellent permselectivity of Nafion.