Two room temperature ionic liquids, 1-butyl-3-methylimidazolium bistriflimide and 1-butyl-3-methylimidazolium nitrate, were employed for enzyme immobilization in a new sol-gel matrix and, for the first time, were successfully applied as electrolyte carriers in a biosensing system. The new sol-gel matrix, based on 3-aminopropyltrimethoxysilane and 1-butyl-3-methylimidazolium bistriflimide mixtures, did not crack even after several weeks when kept dry, and exhibited similar analytical properties to aqueous sol-gel based glucose biosensors. The linear range was up to 1.1 mM of glucose, sensitivity was 62 nA mM−1 and the limit of detection was 28.8 μM. The optimum ionic liquid electrolyte carrier was found to be 1-butyl-3-methylimidazolium nitrate, where the biosensor was made by electrodeposition of the redox mediator, poly(neutral red), and the enzyme was immobilized by cross-linking with glutaraldehyde. The results showed that application of room temperature ionic liquids to biosensors is very promising and can be further exploited.