Here we developed a novel hybrid bipolar electrode (BPE)-electrochemiluminescence (ECL) biosensor based on hybrid bipolar electrode (BPE) for the measurement of cancer cell surface protein using ferrocence (Fc) labeled aptamer as signal recognition and amplification probe. According to the electric neutrality of BPE, the cathode of U-shaped ITO BPE was electrochemically deposited by Au nanoparticles (NPs) to enhance its conductivity and surface area, decrease the overpotential of O2 reduction, which would correspondingly increase the oxidation current of Ru(bpy)3(2+)/tripropylamine (TPA) on the anode of BPE and resulting a ∼4-fold enhancement of ECL intensity. Then a signal amplification strategy was designed by introducing Fc modified aptamer on the anode surface of BPE through hybridization for detecting the amount of mucin-1 on MCF-7 cells. The presence of Fc could not only inhibit the oxidation of Ru(bpy)3(2+) because of its lower oxidation potential, its oxidation product Fc(+) could also quench the ECL of Ru(bpy)3(2+)/TPA by efficient energy-transfer from the excited-state Ru(bpy)3(2+)* to Fc(+), making the ECL intensity greatly quenched. On the basis of the cathodic Au NPs induced ECL enhancing coupled with anodic Fc induced signal quenching amplification, the approach allowed detection of mucin-1 aptamer at a concentration down to 0.5 fM and was capable of detecting a minimum of 20 MCF-7 cells. Besides, the amount of mucin-1 on MCF-7 cells was calculated to be 9041 ± 388 molecules/cell. This approach therefore shows great promise in bioanalysis.