Glucose detection is particularly important for clinical diagnosis and personal prevention and control. Herein, the smartphone-based amperometric glucose sensors were constructed using the NiS/CuS nanorods (NRs) as sensing electrodes. The NiS/CuS NRs were prepared through a facile hydrothermal process accompanied by the subsequent vulcanization treatment. The morphological and structural properties of NiS/CuS NRs were characterized with SEM, EDS, XRD, and XPS. Electrochemical measurements including cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy display that NiS/CuS NRs can act as highly efficient electrocatalyst for glucose detection. The NiS/CuS NRs electrodes present a wide detection range of 1–8000 μM for glucose sensing with the sensitivity of 956.38 μA·mM<sup>−1</sup>·cm<sup>−2</sup>. The detection limit was 0.35 μM (S/N=3). When employed in smartphone-based glucose sensing device, they also display a high sensitivity of 738.09 μA·mM<sup>−1</sup>·cm<sup>−2</sup> and low detection limit of 1.67 μM. Moreover, the smartphone-based glucose sensing device also presents favorable feasibility in determination of glucose in serum samples with the recoveries ranging between 99.5 and 105.8%. The results may provide a promising viewpoint to design other new portable glucose sensors.