Glycans play an important role in various physiological and pathological processes. Metabolic labeling with bioorthogonal chemistry is a distinguished tool for detecting and tracking glycans in cells and in vivo. However, most of the currently available bioorthogonal turn-on probes based on organic fluorophores still suffer from some inevitable deficiencies, including shallow tissue penetration and spontaneous fluorescence. Herein, we designed and reported a bioorthogonal turn-on nanoprobe UCNP-T, which could realize the specific labeling and visualization of glycans on living cell membranes. UCNP-T was constructed based on a multi-spectral upconversion nanophosphor (UCNP) as the luminescence resonance energy transfer (LRET) donor and an organic molecule, tetrazine, as the acceptor. Using the as-prepared UCNP-T, we could specifically label the cell-surface glycans and monitor their level in living mice in real time through the ratio of upconversion luminescence (UCL) emissions of 540 nm to 650 nm (UCL₅₄₀/UCL₆₅₀), providing sensing with highly intrinsic reliability by self-calibration. Thus, the nanoprobe would provide a reliable tool for elucidating the role of glycosylation in cells and in vivo.