A carbon ceramic electrode was modified with a thin film composed of overoxidized polypyrrole, CuO and multi-walled carbon nanotubes. The surface morphology, electrochemical properties and electrocatalytic activity towards the oxidation of glucose of the modified electrode were studied in detail. Benefiting from the high electrocatalytic activity of CuO, the selectivity of OPpy film, and the fast electron transfer rate promoted by MWCNTs, this modified electrode displays good stability, selectivity, high electrocatalytic activity and a low detection limit for the determination of glucose in pH 13 solution. Under the optimum conditions, the linear range for the determination of glucose by cyclic voltammetry is from 20 μM to 10 mM, and the detection limit is 4.0 μM (at an SNR of 3). The amperometric calibration plot covers the 0.20 μM to 2.0 mM concentration range, and the detection limit is 50 nM. The highest sensitivity for the determination of glucose is 3922.6 μA mM−1 cm2.