A sensitive and selective electrochemical method for the determination of norepinephrine (NE) was developed by using a calix[4]arene crown-4 ether (CACE) film modified glassy carbon electrode (GCE). The fabrication of the CACE film and its electrocatalytic effect for electrochemical oxidation of NE were investigated by electrochemical impedance spectroscopy (EIS) and voltammetric methods. It was found that the electrochemical behavior of NE on the CACE film modified electrode depended on the film thickness of CACE. Based on the electrochemical data, the CACE film surface coverage (theta), the charge transfer number (z) and the amount of the surface-bound NE (Gamma) were calculated. The effects of the experimental variables such as the solution pH and the applied potential were investigated for optimum analytical performance. Excellent linear relationships were obtained in two concentration ranges of NE: one was from 0.55 to 9.7 microM and the other was 9.7 to 230 microM. The detection limit (S/N = 3) of NE obtained by steady-state amperometry was 0.28 microM. The relative standard deviation (RSD) for 10 successive measurements of 0.05 mM NE was 2.2%. This method could be applied for the direct determination of NE in injection samples. In addition, effects of possible interferences (e.g. ascorbic acid, AA) were investigated. The present work provides an effective method for amperometric determination of NE by using a CACE modified electrode.