Exosomal programmed cell death ligand 1 (exoPD-L1) has emerged as a promising biomarker for cancer diagnosis and immunotherapy outcome prediction. However, the existing quantitation methods are incapable of addressing the heterogeneity of exoPD-L1 glycosylation, which has been demonstrated to be the institutional basis for PD-L1/PD-1 interaction and the crucial participant in inhibiting the activity of CD8+ T cells. Herein, we developed an aptamer and lectin-induced proximity ligation assay (PLA) combined with quantitative real-time PCR for precise quantitation of glycosylated exoPD-L1. Leveraging the metabolism-free lectin labeling of glycosylation, the glycosylation-independent aptamer tagging of PD-L1, and excellent selectivity of dual-recognition, this method enables glycosylated exoPD-L1 quantitation with high sensitivity and selectivity in a wash-free manner. As a result, this method is able to distinguish the levels of glycosylated exoPD-L1 between healthy donors and cancer patients with sensitivity and specificity of 100%. Compared with the total circulating exoPD-L1 level, glycosylated exoPD-L1 was for the first time identified to be a more reliable biomarker for tumor diagnosis. Overall, this strategy holds a great potential for revealing the significance of exoPD-L1 glycosylation and converting glycosylated exoPD-L1 into a reliable clinical indicator.