The rapid and accurate assessment of glucose concentration has been demonstrated to play a significant role in human health, such as the diagnosis and treatment of diabetes, pharmaceutical research and quality monitoring in the food industry, necessitating further development of the performance for glucose sensor especially at low concentrations. However, glucose oxidase-based sensors suffer from crucial restriction in bioactivity because of their poor environmental tolerance. Recently, catalytic nanomaterials with enzyme-mimicking activity, known as nanozymes, have gained considerable interest to overcome the drawback. In this scenario, we report an inspiring surface plasmon resonance (SPR) sensor for non-enzymatic glucose detection employing ZnO nanoparticles and MoSe2 nanosheets composite (MoSe2/ZnO) as sensing film, featuring desirable advantages of high sensitivity and selectivity, lab-free and low cost. The ZnO was used to specifically recognize and bind glucose, and further signal amplification was realized by incorporating of MoSe2 owing to its larger specific surface area and favorable bio-compatibility, as well as high electron mobility. These unique features of MoSe2/ZnO composite film result in an obvious improvement of sensitivity for glucose detection. Experimental results show that the measurement sensitivity of the proposed sensor could reach 72.17 nm/(mg/mL) and a detection limit of 4.16 μg/mL by appropriately optimizing the componential constitutions of MoSe2/ZnO composite. In addition, the favorable selectivity, repeatability and stability are demonstrated as well. This facile and cost-effective work provides a novel strategy for constructing high-performance SPR sensor for glucose detection and a prospective application in biomedicine and human health monitoring.