Immune checkpoint therapy has provided a weapon against cancer, but its response rate has been extremely low due to the lack of effective predictors. Herein, we developed a FRET strategy based on lectin for glycan labelling and an aptamer for PD-L1 antigen recognition for visualization of PD-L1-specific glycosylation (FLAG). The FLAG strategy combines the PD-L1 aptamer, which efficiently labels PD-L1 polyantigen with smaller steric hindrance than PD-L1 antibody, and metabolism-free lectin labelling for glycosylation. As a result, the FLAG strategy enables in situ visualization of PD-L1-specific glycosylation on the tissue section while maintaining the spatial context and tissue architecture. Due to non-metabolic labelling, the FLAG strategy revealed that the tissue level of PD-L1-specific glycosylation is correlated with the efficacy of PD-1/PD-L1 therapy. Overall, the FLAG strategy provides a powerful tool for revealing the significance of PD-L1 glycosylation, offering the unprecedented potential for immunophenotypic differential analysis to predict the immunotherapy response.