Abstract A novel carbon nanotube-modified glassy carbon electrode was described for the direct determination of pyridoxine. The electrochemical behavior of pyridoxine was investigated, and a well-defined oxidation peak with high sensitivity was observed at the modified electrode. Owing to the unique structure and extraordinary properties of multi-wall carbon nanotube (MWNT), the MWNT-modified glassy carbon electrode shows obvious electrocatalytic activity to the oxidation of pyridoxine, since it greatly enhances the oxidation peak current of pyridoxine as well as lowers its oxidation overpotential. Based on this, a very sensitive and simple voltammetric method was developed for the measurement of pyridoxine. A sensitive linear voltammetric response for pyridoxine was obtained in the concentration range of 5 × 10 −7–1 × 10 −4 mol/L, and the detection limit is 2 × 10 −7 mol/L using differential pulse voltammetry. Compared with other voltammetric methods, this proposed method possesses many advantages such as very low detection limit, fast response, low cost and simplicity. The practical application of this new analytical method was demonstrated with pyridoxine drugs.