A novel electrochemical glucose biosensor was developed based on in situ covalent immobilization of glucose oxidase (GOx) by one-pot chitosan (CS)-incorporated sol-gel process in the presence of Prussian blue deposited multi-walled carbon nanotubes hybrids (PB/MWNTs) using 3-isocyanatopropyltriethoxysilane (ICPTES) as both a sol-gel precursor and a covalent coupling agent for GOx and CS. The electrode modified with the PB/MWNTs-GOx-CS-ICPTES sol-gel composite film showed good electrical conductivity and effective low-potential electron transfer mediation toward H2O2 reduction attributed to the incorporation of PB/MWNTs. The biosensor exhibited a linear response to glucose in the concentration range from 2.5×10(-5) to 1.3×10(-3) M with a correlation coefficient of 0.9998, a detection limit of 7.5×10(-6) M, a low response time (10s ), good sensitivity and high anti-interference ability. Compared with the control biosensor based on the traditional tetraethoxysilane derived sol-gel composite film, the biosensor showed a similarly small apparent Michaelis-Menten constant of 3.67 mM but much higher electrochemical and biosensing stability.