Exosomal glycoproteins play important roles in many physiological and pathological functions. However, the existing methods for studying glycosylation of exosomal proteins of interest are often cumbersome and affect the exosome integrity. Herein, we developed a dual labeling strategy based on a protein-specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes. The glycosylation of exosomal PD-L1 (exoPD-L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD-L1 aptamers bound on exoPD-L1 and fluorescent tags on glycans introduced via metabolic glycan labeling. This method enables in situ visualization and biological function study of exosomal protein glycosylation. Through this strategy, exoPD-L1 glycosylation was confirmed for the first time to be required in interaction with PD-1 and participated in inhibiting of CD8+ T cell proliferation. In general, we have developed an efficient and non-destructive method to study the presence and function of exosomal protein-specific glycosylation in situ, which provides a powerful tool for exosomal glycoproteomics research.