Tumor-derived exosomes carry lots of tumor-specific proteins, attracting more attention as ideal biomarkers for non-invasive diagnosis. However, due to the low concentration of exosomes in biological samples, the development of an ultrasensitive method for tumor-specific exosomal protein detection is highly desired. Here, a sensitive Single Microbead (MB)-based Aptasensor coupled with Terminal deoxynucleotidyl transferase (TdT)triggered signal amplification (SMAT) is reported to detect tumor-specific exosomal proteins. In the SMAT detection system, a single MB is used as the reaction carrier and firstly modified by the antibodies of generic membrane biomarkers (anti-CD63 and anti-CD81) of exosomes for efficient enrichment of exosomes in samples. Then, the DNA aptamers immobilized on gold nanoparticles (AuNPs) are applied to specifically bind to the tumor-specific proteins on exosome surfaces. Subsequently, the DNA aptamers can be efficiently extended by TdT to generate a long poly(dT) tail. By hybridization with lots of fluorescein-labeled poly(dA)25 (FAM-poly(dA)25) probes, large amounts of fluorescence molecules will be highly enriched on one MB and in situ monitored with fluorescence imaging. With this strategy, exosomal EpCAM protein (Exo-EpCAM) has been sensitively detected from the samples containing 55 particles/mu L tumor-derived exosomes. Furthermore, the proposed method could be applied for detection of Exo-EpCAM from plasma microsamples, indicating great application prospect in future for clinical diagnosis.