A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H(2)O(2)) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H(2)O(2) and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H(2)O(2) ranged from 2.0 x 10(-7) to 4.6 x 10(-5)moll(-1) with a detection limit of 8.1 x 10(-8)moll(-1). The apparent Michaelis-Menten constant was determined to be 45.18micromoll(-1). Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.