Tumor-derived exosomalproteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is severely hindered by large numbers of normal cell- derived exosomes. Herein, we developed a dual-target-specific aptamer recognition activated in-situ connection system on exosome membrane combined with droplet digitalPCR (ddPCR) (TRACER) for precise quantitation of tumor-derived exosomal PD-L1 (Exo-_PD-L1). Taking advantage of the high binding affinity of aptamers, excellent selectivity of dual-aptamer recognition and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor-derivedExo-_PD-L1 in a wash-free manner. Due to the excellent sensitivity, the level of tumor-derived Exo-_PD-L1 detected by TRACER can effectively distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo-_PD-L1. Overall, TRACER strategy holds great potential for analysis of different subtypes of exosomes, unprecedented capability for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.