Glucose detection using sensing materials has lately received interest due to the increased demand for sensitive and selective glucose sensors in pharmaceutical, clinical, and industrial settings. Carbon nanotubes (CNTs) are used intensively as a specific class of effective electrode substances in electrochemical sensing due to their large surface area and interesting physical and electrochemical characteristics. Nickel is an attractive transition metal for glucose electrooxidation with high catalytic activity. In this study, CNT/MoS2/NiNPs nanocomposites with different CNT/MoS2 ratios have been prepared by a hydrothermal reaction. The CNT/MoS2 nanocomposites were characterized by X-ray diffraction (XRD), Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR), and their morphology and composition were characterized by field emission scanning electron microscopy (FESEM). Electrocatalytic activity of the as-prepared nanomaterials towards glucose oxidation was investigated by cyclic voltammetry and amperometry in alkaline media. An excellent sensitivity value of 1212 μA·mM−1·cm−2 with a wide linear range (0.05–0.65 mM), a low detection threshold of 0.197 μM and a short response time (3 s) were achieved by the hybrid CNT/MoS2/NiNPs sensor. Its superior catalytic activity and low cost make this hybrid very promising for applications in the direct sensing of glucose.