Silica xerogel was widely used in the development of biosensors by coupling the desired biological components to various transducers. Unfortunately, the application of xerogels is limited due to their poor mechanical properties and poor structural maintenance of entrapped biomaterials. In this study, the availability of poly(dimethylsiloxane) (PDMS)-modified silica sol-gel (TEOS/PDMS Ormosil) glass in the immobilization of enzymes and its application in the fabrication of amperometric biosensor was investigated. We demonstrated that most of activity of encapsulated horseradish peroxidase can be preserved in PDMS-modified SiO2 glass compared with conventional silica xerogel. The synthesized monoliths exhibited high transparency and crack-free. The enzyme electrode based on glucose oxidase encapsulated TEOS/PDMS Ormosil glass on Pd electrode was fabricated and characterized electrochemically. The characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The biosensor exhibited a series of good properties: high sensitivity (1.63 μA mM-1 analyte), short response time and high reproducibility. The results indicate that TEOS/PDMS Ormosil glass has great potential to the immobilization of biomaterials as well as the fabrication of biosensors.