The amino acid glutamate is the major excitatory neurotransmitter used in the nervous system for interneuronal communication. It is used throughout the brain by various neuronal pathways including those involved in learning and memory, locomotion, and sensory perception. Because glutamate is released from neurons on a millisecond time scale into sub-micrometer spaces, the development of a glutamate biosensor with high temporal and spatial resolution is of great interest for the study of neurological function and disease. Here, we demonstrate the feasibility of an optical glutamate sensor based on the sol-gel encapsulation of the enzyme glutamate dehydrogenase (GDH). GDH catalyses the oxidative deamination of glutamate and the reduction of NAD+ to NADH. NADH fluorescence is the basis of the sensor detection. Thermodynamic and kinetic studies show that GDH remains active in the sol-gel matrix and that the reaction rate is correlated to the glutamate concentration.