Glycans exhibit pronounced spatial heterogeneity in tissues and organs. However, glycan detection in these complex biological systems remains challenged by strong background interference, limited labeling efficiency, and difficulty in faithfully preserving spatial information. To address these issues, we developed a reduction-assisted galactose oxidase labeling platform (ReGOL). By introducing NaBH4 prereduction to suppress fixation-derived background and integrating galactose oxidase oxidation with fast hydrazone ligation into an efficient readout workflow, ReGOL enables high-fidelity imaging of terminal galactose/N-acetylgalactosamine (Gal/GalNAc) in tissue sections. The method is applicable to both frozen and paraffin-embedded sections and can be sequentially combined with hematoxylin and eosin (H&E) staining on the same section, allowing direct correlation between glycan signals and histological structures. In addition, we established a neuraminidase-ReGOL versus ReGOL comparative strategy to distinguish exposed terminal Gal/GalNAc from sialic acid-capped counterparts, and applied it to analyze changes in terminal glycan spatial distribution and glomerular morphology in a renal fibrosis model. In a bladder cancer model, ReGOL clearly delineated the tumor-peritumoral-urothelium boundary and revealed tumor-associated glycan heterogeneity. Building on this, we further translated the 15 min labeling workflow to the bladder in vivo, enabling selective glycan imaging of tumor regions. This system also enabled selective installation of bioorthogonal chemical handles onto the glycocalyx of bladder tumors in vivo, thereby supporting subsequent chemical functionalization. Collectively, this work establishes an oxidation-based glycan analysis platform applicable to both tissue sections and living systems, and provides a practical method for spatial analysis and localized chemical intervention of terminal glycans in complex biological environments.