An effective two-step synthesis route was developed to obtain a heteroatom-enriched activated carbon nickel oxide (HAC-NiO) nanocomposite by an eco-friendly chemical activation and hydrothermal method. The nanomaterials were characterized by scanning electron microscopy, X-ray diffraction, N₂ adsorption desorption isotherms. Furthermore, the NiO-HAC nanocomposite was used to modify glass carbon electrode (NiO-HAC/GCE) and build a novel glucose sensor. The electrochemical properties revealed that it exhibited a wide linear range (10 μM-3.3 mM) and a low detection limit (1 μM) toward glucose oxidation. The excellent electrocatalytic performance can be attributed to the unique surface architectures of NiO with good electron transfer rate and HAC with large active surface area during the electrochemical sensing process. High reproducibility, good anti-interference, desirable stability and enough precision made it a potential material for the development of enzyme-free sensor in real sample analysis.