A novel and sensitive electrochemiluminescence (ECL) method for ethanol biosensor was developed by co‐immobilizing the enzyme and ECL reagent Ru(bpy)32+ on the poly‐(3,4‐ethylene dioxythiophene) and polystyrene sulfonate functionalized graphene (PEDOT‐PSS‐G) nanocomposite film. Positively charged Ru(bpy)32+ could be immobilized effectively on the electrode surface with the negatively charged PSS and graphene, which provided a stable ECL platform for further modification with the enzyme. Moreover, the introduction of PEDOT and graphene can be acted as a conducting pathway to accelerate the electron transfer due to the high conductivity. Such biosensor combined enzymatic selectivity with the amplification of PEDOT‐PSS‐G performed well with a wide linear range, high sensitivity and good stability. The sensing platform was successfully applied to determine the amounts of alcohol in real samples.