An improved amperometric glucose biosensor based on glucose oxidase immobilized in sol-gel chitosan/silica hybrid composite film, which was prepared from chitosan (CS) and methyltrimethoxysilane (MTOS), on the surface of Prussian blue (PB)-modified glass carbon electrode was developed. The film was characterized by FT-IR. Effects of some experimental variables such as ratio of CS to silica, buffer pH, temperature, and applied potential on the current response of the biosensor were investigated. The biosensor fabricated under optimal conditions had a linear response to glucose over the range 5.0 x 10(-5) to 2.6 x 10(-2) M with a correlation coefficient of 0.9948 and a detection limit of 8.0 x 10(-6) M based on S/N = 3. The biosensor had a fast response time of less than 10 s, a high sensitivity of 420 nA mM(-1), a long-term stability of over 60 days, and a good selectivity. The apparent Michaelis-Menten constant K(m) was found to be 3.2 x 10(-3) M. The activation energy for enzymatic reaction was calculated to be 21.9 kJ mol(-1). This method has been used to determine the glucose concentration in real human blood samples.