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Core fucosylation, the attachment of α1,6-fucose to the innermost GlcNAc residue of N-glycans, is a unique protein glycosylation in living organisms. It plays significant roles in diverse physiological and pathological processes, and its upregulation has been observed in many cancers. Core fucosylation has a strong relationship with tumor growth, inva-sion, metastasis, prognosis, and immune evasion. Yet, the details are still largely unknown due to the lack of an effec-tive analytical method. Here, a site-specific and reversible labeling strategy for rapid and sensitive probing core fucosyla-tion was developed. Taking the newly developed strategy, core-fucosylated proteins located on the cell-surface were selectively labeled and analyzed. The profile of cell-surface glycoproteins provides an in-depth understanding of the biological functions of core fucosylation.