In this study, a low‐cost, disposable, sensitive and selective electrochemical sensor was developed for glucose determination, which is based on a pencil graphite electrode (PGE) modified with nickel hydroxide. Cyclic voltammetry was used to deposit nickel on PGE. The morphology and composition of the unmodified and modified PGE electrodes were characterized by field‐emission scanning electron microscopy (FE‐SEM), atomic force microscopy (AFM) and energy‐dispersive X‐ray spectroscopy (EDX). The performance of the as modified electrode towards the electrooxidation of glucose in alkaline media was evaluated with cyclic voltammetry and amperometry techniques. A linear relationship between the concentration of glucose and oxidation peak currents was observed in two concentration ranges of 0.004–3.5 mM and 3.5–9.0 mM. The detection limit was 2 μM at signal‐to‐noise ratio of 3. In addition to a fast response time (<2 s), the proposed sensor preserved 93 % of its original response towards glucose after 28 days. Also, the sensor showed a good reproducibility and high selectivity for glucose oxidation in the presence of common interfering species. The use of Ni(OH)2/PGE sensor was successfully tested in the determination of glucose in human blood serum for the first time in the literature.