In this study, a copper (II) oxide‐glutardialdehyde modified boron doped diamond electrode was used as a sensor for the electroanalysis of pyridoxine hydrochloride (vitamin B6) for the first time. A copper oxide‐glutardialdehyde modified boron doped diamond electrode (CuO‐GA2/BDDE) was prepared by a simple drop casting method on boron diamond electrode (BDDE) by suspending CuO with glutardialdehyde (CuO‐GA2). The electrochemical behavior of vitamin B6 was examined using cyclic voltammetry (CV) and its electrochemical determination was found by differential pulse voltammetry (DPV). Under optimum experimental conditions, the linear working range of vitamin B6 on CuO‐GA2/BDDE via the DPV method was calculated as 5.96×10−6–3.75×10−4 mol.L−1. The limit of detection (LOD) was calculated as 1.99×10−7 mol.L−1. In order to demonstrate the selectivity of the method improved for the quantitative determination of vitamin B6 on the CuO‐GA2/BDDE, the interference effect of some cations (Zn2+, Cu2+, Pb2+) and anions (NO3−, Cl−) was investigated via the DPV method. The improved electrode was successfully used for the electroanalysis of vitamin B6 in a tablet sample of the commercial Tribeksol® (deva) (250/250/1 mg B1+B6+B12).