Exosomal glycoproteins play significant roles in many physiological and pathological procedures. However, the current methods for studying exosomal glycoproteins have low sensitivity or can affect exosomal biological function. Herein, we developed a proximity dual-tagging strategy using an induced hybridization chain reaction (HCR) from the target’s non-functional epitope for amplified visualization and functional exploration of exosomal protein-specific glycosylation. This strategy leverages dual-tagging based on the aptamer with little influence on target function and metabolic glycan labelling, and the rigid product and high sensitivity of HCR. The method improves the signal of visualizing exosomal PD-L1 (exoPD-L1) by 7.7-fold compared to the signal without HCR amplification without affecting the natural exoPD-L1/PD-1 interaction. As a result, we verified that the interaction between exoPD-L1 and PD-1 positive cells is positively correlated to the glycosylation level of exoPD-L1. Overall, we have developed a sensitive method with little functional influence to visualize exosomal protein-specific glycosylation in situ, offering a powerful tool for studying the biological implications of exosomal glycoproteins.