Through a simple method, a new kind of Mn3O4 nanoparticles (Mn3O4NPs) decorated nitrogen-doped graphene (N-GR) was synthesized firstly, and then a Mn3O4NPs/N-GR modified carbon paste electrode (CPE) was fabricated. Several techniques, such as X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and electrochemistry, were performed to characterize Mn3O4NPs/N-GR. A large amount of N on the graphene surface (about 9.86% of atomic ratio) was detected by XPS. Mn3O4 nanoparticles (50–80nm) homogeneously coated on N-GR. Mn3O4NPs/N-GR/CPE was used for sensitive non-enzymatic glucose sensing, which exhibited the response current of glucose ca. 10-fold higher than that of pure CPE. Under the optimum determination conditions, Mn3O4NPs/N-GR/CPE provided glucose with a broader detection range of 2.5–529.5μM with a low detection limit (1.0μM, S/N=3). This novel method was successfully used to determine glucose in human urine sample.