In this work, we report a new sensing approach based on electrogenerated chemiluminescence (ECL) in an electrodeposited redox hydrogel using glucose dehydrogenase as a model system. The ECL-hydrogel films were electrodeposited by potential cycling of a PBS solution containing [poly(4-vinylpyridine)Ru(2,2'-bipyridine)2Cl-]+/2+. The film was easily prepared in a rapid, reproducible and well-controlled one-step procedure. The deposited hydrogel film is permeable to water-soluble chemicals and biochemicals, like enzyme substrates and coenzymes. Electrochemistry and ECL of NADH were studied at the level of the hydrogel film.Then we showed that the intensity of the ECL of NADH produced by the enzymatic activity varies with the enzyme substrate concentration. Such sensing approach combines enzymatic selectivity with the ECL advantages at low oxidation potential.