An amperometric biosensor for hydrogen peroxide (11201) was fabricated based on immobilization of hemoglobin (Hb) on DNA/Poly-2,6-pyridinediamine (PPD) modified Au electrode. PPD thin films were firstly electro-deposited on Au electrode surface which provide a template to attach negatively charged DNA molecules by electrostatic attraction. The adsorbed DNA network provides a good microenvironment for the immobilization of biomolecules and promotes electron transfer between the immobilized Hb and the electrode surface. The fabrication process of the biosensor was characterized by electrochemical impedance spectroscopy. Experimental conditions influencing the biosensor performance such as pH, potential and temperature were assessed and optimized. The proposed biosensor displayed a good electrocatalytic response to the reduction of H2O2 its linear range is 1.7 mu M to 3 mM with a detection limit of 1.0 mu M based on the signal-to-noise ratio of 3 (S/N= 3) under the optimized conditions. The Michaelis-Menten constant K-m(app) of Hb immobilized on the electrode surface was found to be 0.8 mM. The biosensor shows high sensitivity and stability. Importantly, this deposition methodology could be further developed for the immobilization of other proteins and biocompounds. (c) 2007 Elsevier B.V All rights reserved.