A one-spot method was developed for the synthesis of graphene sheet decorated with copper nanoparticles using different reduction temperatures via a molecular level mixing process. Here, we demonstrate that the reduction temperature is a crucial determinant of the properties of reduced graphene oxide (RGO)/metal composite and its electrocatalytic application in glucose sensing. To show this, we prepared a series of RGO/Cu composites at different reduction temperatures and examined the change rules of size, loading and dispersion of Cu particles, and the reduction extent of the RGO. Results showed that the Cu particle size increased with increasing reduction temperatures due to the Ostwald ripening process. Meanwhile, the Cu loading decreased with increasing reduction temperatures and the aggregation had not appeared in the high Cu loading situation. Additionally, the increasing reduction temperatures led to the decreasing concentrations of various oxygen-containing functional group of RGO with various degrees. The cyclic voltammogram showed that the RGO/metal composites fabricated under lower reduction temperatures exhibited higher electrocatalytic activity for glucose sensing, which was attributed to the higher surface area from larger loading of RGO/metal composites with smaller particle size. It can be concluded that the above factors play more significant roles in electrocatalytic efficiency than the decreased electron transfer rate between RGO and Cu within a certain range. These results highlight the importance of the reduction temperature influencing the properties of the RGO/metal composite and its application. We believe that these findings can be of great value in the further developing RGO/metal-based sensors for electrochemical detection of different analytes in emerging fields.