Choline (Ch) and acetylcholine (ACh) modified glassy carbon electrodes have been prepared and characterized by X-ray photoelectron spectroscopy (XPS), UV-visible spectroelectrochemistry (LTV-Vis), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The modification of ACh was that the Ch was covalently bounded on the electrode surface accompanied by acetate anions embedded and adsorbed by electrostatics in the Ch modified layer, forming a novel choline/acetate interdigitate assembly. This provided an excellent example of a mixed two-component modification from a native acetylcholine molecule on a carbon electrode. These two modified electrodes were applied to the electrocatalytic oxidation of dopamine (DA), serotonin (5-HT) and ascorbic acid (AA), and resolved the overlapping of the anodic peaks of DA, 5-HT and AA into three well-defined voltammetric peaks in CV or different pulse voltammetry (DPV). This can be used for simultaneous determination of these species in a mixture. The stability of the acetate/choline/glass carbon electrode (ACh/GCE) sensing system was good, with up to at least 30 days of continual operation. (C) 2004 Elsevier B.V. All rights reserved.