Tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy) 3 2+) and dehydrogenase enzymes are immobilized in cation exchange polymer films to create electrogenerated chemiluminescence (ECL) biosensors. Eastman AQ and Nafion polymers were used as immobilization support in a regenerable ECL sensor for NADH and NADPH and in biosensors for glucose, lactate and ethanol. The biosensor design places an enzyme-loaded polymer film adjacent to a Ru(bpy) 3 2+-loaded polymer film covering a platinum electrode. The modified electrode was used in a flow injection analysis system as an ECL detector. The CL response to samples containing enzyme substrate and cofactor (NAD +) result from the Ru(bpy) 3 2+ ECL reaction with NADH produced by the enzyme. The analytical response to NADH using an AQ film modified with Ru(bpy) 3 2+. The coimmobilization of that seen for Nafion films modified with Ru(bpy) 3 2+. Teh coimmnolization Ru(bpy) 3 2+ and dehydrogenase enzymes in the same film showed reduced enzyme activity which led to various biosensor designs with adjacent layers. The ECL biosensor concept was shown applicable for both NAD +-and NADP +-dependent enzymes, where NADPH detection ranged from 0·01 to 100 μM NADPH.