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 drying behavior and poor mechanical properties. ORMOSILs, organically modified ceramic materials, are synthesized using sol-gel techniques and permit the fabrication of a wide variety of compositions as thick coatings or monoliths. In this study the availability of poly(dimethylsiloxane) (PDMS)-modified ORMOSIL in the immobilization of enzymes as well as its application in the fabrication of amperometric biosensor was investigated. We demonstrated that the stability of encapsulated horseradish peroxidase can be greatly enhanced in TEOS/PDMS ORMOSIL compared with TEOS xerogel. The synthesized monoliths exhibited high transparency and crack-free. The enzyme electrode based on glucose oxidase encapsulated ORMOSIL on Pd electrode was fabricated and characterized electrochemically. The results indicate that TEOS/PDMS ORMOSIL has a great potential for the immobilization of biological molecules as well as the fabrication of biosensors.