This study investigated the response of different-age biofilms developed on membrane surface to a chemical uncoupler 3, 3', 4', 5-tetrachlorosalicylanilide (TCS). Results showed that adenosine triphosphate (ATP) dissipation caused by TCS would promote different-age biofilms detachment, whereas chemically inhibited cellular ATP synthesis subsequently suppressed autoinducer-2 (AI-2) and extracellular polymeric substances (EPS) production. The extent of biofilm detachment was found to be closely related to AI-2-regulated EPS contents of bacteria. It was revealed that energy dissipation induced biofilm detachability was controlled by AI-2 regulated cellular communication via AI-2-mediated EPS secretion. This study would lead to a new cleaning strategy of biologically fouled membrane.