A nonenzymatic glucose sensor was constructed by electrodepositing molybdenum sulfide (MoS x )-nickel (II) hydroxide (Ni(OH) 2 ) in sequence on a flexible carbon nanotube/polyimide (CNT/PI) composite membrane. The sensing material was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The electrocatalytic activity of the as-prepared nanomaterial toward glucose oxidation was investigated by cyclic voltammetry and amperometric measurement. The Ni(OH) 2 /MoS x /CNT/PI sensor demonstrated excellent properties including a wide linear range from 10 to 1600 μM of glucose, rapid response (<3 s), low detection limit of 5.4 μM, good selectivity, good repeatability, and long-term stability (2 weeks). The superior performances were attributed to the pronounced synergistic effect between Ni(OH) 2 and MoS x . Furthermore, the excellent sensor was successfully applied to detect glucose in human blood serum samples by standard addition method with satisfactory recovery.