Well-defined single-atom catalysts (SACs) serve as ideal model systems for directly comparing experimental results with theoretical calculations, offering profound insights into heterogeneous catalytic processes. However, precisely designing and controllably synthesizing SACs remain challenging due to the unpredictable structure evolution of active sites and generation of embedded active sites, which may bring about steric hindrance during chemical reactions. Herein, we present the precious nonpyrolysis synthesis of Re SACs with a well-defined phenanthroline coordination supported by NiO (Re1-phen/NiO). Multiple experimental characterizations together with theoretical calculations unravel the idea that the isolated Re atoms are suspended on the NiO surface, connected by phenanthroline ligands standing perpendicular to the surface. This unique structure provides the Re1-phen/NiO SAC with a strong capability to activate glucose molecules, enabling fully exposed Re=O double bonds in an open-ended reaction environment to simultaneously react with hydroxyl and aldehyde groups at both ends of the glucose molecule, rapidly forming glucaric acid.