A new strategy for fabricating a sensitivity-enhanced glucose biosensor was presented, based on multi-walled carbon nanotubes (CNT), Pt nanoparticles (PtNP) and sol-gel of chitosan (CS)/silica organic-inorganic hybrid composite. PtNP-CS solution was synthesized through the reduction of PtCl(6)(2-) by NaBH(4) at room temperature. Benefited from the amino groups of CS, a stable PtNP gel was obtained, and a CNT-PtNP-CS solution was prepared by dispersing CNT functionalized with carboxylic groups in PtNP-CS solution. The CS/silica hybrid sol-gel was produced by mixing methyltrimethoxysilane (MTOS) with the CNT-PtNP-CS solution. Then, with the immobilization of glucose oxidase (GOD) into the sol-gel, the glucose biosensor of GOD-CNT-PtNP-CS-MTOS-GCE was fabricated. The properties of resulting glucose biosensor were measured by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In phosphate buffer solutions (PBS, pH 6.8), nearly interference free determination of glucose was realized at low applied potential of 0.1V, with a wide linear range of 1.2x10(-6) to 6.0x10(-3)M, low detection limit of 3.0x10(-7)M, high sensitivity of 2.08microA mM(-1), and a fast response time (within 5s). The results showed that the biosensor provided the high synergistic electrocatalytic action, and exhibited good reproducibility, long-term stability. Subsequently, the novel biosensor was applied for the determination of glucose in human serum sample, and good recovery was obtained (in the range of 95-104%).