A water-soluble chitosan derivation O-carboxymethyl chitosan (CMCS) was developed for the efficient immobilization of horseradish peroxidase (HRP) to construct a H2O2 biosensor. Fourier Transform Infrared (FT-IR) spectrophotometer, Atomic Force Microscope (AFM) and electrochemical method were used to characterize the biosensor. The biosensor was used to determine hydrogen peroxide (H2O2) quantitatively in the presence of methylene blue which was used as a mediator to transfer electrons between the electrode and HRP. It offered a linear range of 2.0 x 10(-6) -3.7 x 10(-5) and 4.2 x 10(-5)- 4.57 x 10(-4) M, with a detection limit of 5.32 x 10(-7) M estimated at a signal-to-noise ration of 3. The apparent Michaelis-Menten constant (K-M(app)) of the biosensor was 3.544 x 10(-5) M. (C) 2005 Elsevier B.V. All rights reserved.