A new type of sol /gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H2O2 biosensor. The composite filmwas used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By usingfluorescent label, a protein density on sol /gel/chitosan has been calculated to be 3.14×1012 molecules cm 2. With the aid ofcatechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0×10-9-1.0×10-7 mol-1 and adetection limit of 2×10-9 mol-1. Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant KappM is found to be 1.30 mmol 1. The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol 1.The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4℃.