Exosomal surface glycan reveals the biological function and molecular information on the protein, especially in indicating the pathogenesis of certain diseases through monitoring of specific protein glycosylation accurately. However, in situ and nondestructive measurement techniques for certain Exosomal glycoproteins are still lacking. In this work, combined with on-chip purification, we designed a proximity ligation assay-induced rolling circle amplification (RCA) strategy for highly sensitive identification of Exosomal protein-specific glycosylation based on a couple of proximity probes to target Exosomal protein and the protein-specific glycosylation site. Benefiting from efficient separation, scalable dual-recognition, and proximity-triggered RCA amplification, the proposed strategy could convert different protein-specific glycan levels to prominent changes in absorbance signals, resulting in accurate quantification of specific glycosylated Exosomal protein. When detecting the glycosylated PD-L1 on MDA-MB-231 exosomes and glycosylated PTK7 on HepG2 exosomes, the detection limits were calculated to be as low as 1.04 × 104 and 2.759 × 103 particles/mL, respectively. In addition, we further expand the dual-recognition site to investigate the potential correlation of Exosomal glycosylation with polarization of THP-1 cells toward the tumor-suppressive M1 phenotype. Overall, this strategy provides a universal tool for multiple analyses of diverse protein-specific glycosylated exosomes, exhibiting enormous potential to explore exosome function and search for new early diagnosis markers.