Much attention has been paid to immobilization of enzymes to improve enzyme stability and permit its reuse. Glucose oxidase entrapment in different kinds of silica sol-gel matrices was investigated. The enzyme showed stable activity for 11 uses in the sol-gel with tetramethyl orthosilicate (TMOS) as precursor, and at least 7 uses in the sol-gel induced electrochemically with tetraethyl orthosilicate (TEOS) as precursor. The sol-gel made with TEOS as precursor and HCl as catalyst showed stability in enzyme activity for 11 uses but the activity decreases on the same sol-gel when modified with silica nanoparticles. Factors such as optimal incubation time of glucose solution, the reproducibility between different sol-gels, and the storage time were investigated. Good linearity and analytical results on real samples were obtained. The detection method was based on a colorimetric method for determining the concentration of hydrogen peroxide produced from the oxidation reaction of glucose.