An electro-deposition method was adopted to prepare gold-capped pencil graphite surface using pencil graphite electrode(PGE).By chemical bonding of Au-S,L-cysteine(L-Cys) can be stably immobilized onto the surface of gold,followed by an assembling with acridine orange(AO) via the electrostatic interaction between AO and L-cysteine to obtain an AO/L-Cys/Au/PGE multilayer membrane.The prepared multilayer membrane was connected between the terminal joints of the working electrode and the counter electrode,together with a reference electrode,to construct a novel system.The whole multilayer membrane fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy.The interaction of AO and the subsequent introduction of bovine serum albumin(BSA) resulted in a significant change of zero current potential(ΔEzcp) utilizing zero current potentiometry,thus the electrochemical quantification of BSA could thus be realized.The results showed that ΔEzcp and logarithmic of BSA concentration was linear in a wide concentration range of 1.0×10-9-1.0×10-5 mol/L(r=0.997 0),and the detection limit was 1.41×10-11 mol/L.The multilayer membrane exhibited good selectivety and reproducibility,and was applicable for the determiantion of other proteins.