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Tumor-derived extracellular vesicles (TEVs) have emerged as promising sources of diagnostic and prognostic biomarkers for nasopharyngeal carcinoma (NPC). However, the lack of high-sensitivity analytic methods for ultratrace membrane proteins on TEVs hamper their clinical application of TEVs. Herein, by combining aptamers that specifically bind to protein targets on TEVs, PCR-based exponential amplification and CRISPR/Cas12a real-time DNA detection, we developed a novel technique, termed the aptamer-CRISPR/Cas12a assay, to detect CD109+ and EGFR+ TEVs from cell lines and complex biofluids. The platform enables highly sensitive detection of CD109+ and EGFR+ TEVs at as low as 100 particles/mL with a linear range spanning 6 orders of magnitude (102-108 particles/mL), which was found to be sufficient to effectively detect TEV proteins directly in low-volume (50 μl) samples. Furthermore, clinical serum sample analysis verified that the combination of serum CD109+ and EGFR+ TEV levels yielded high diagnostic accuracy, with an AUC of 0.934 (95% CI: 0.868–1.000), a sensitivity of 84.1% and a specificity of 85.0%, in discriminating NPC from healthy controls. Moreover, the dramatic decrease in both biomarkers in responders after radiotherapy indicated their potential roles in radiotherapy surveillance. Given that the aptamer-CRISPR/Cas12a assay rapidly and conveniently detects ultralow concentrations of CD109+ and EGFR+ TEVs directly in serum, it could be useful in NPC diagnosis and prognosis. Image 1 • A novel approach, called the aptamer-CRISPR/Cas12a assay, was developed. • A highly sensitive, specific and cost-effective method to measure EV surface protein. • Serum CD109+ and EGFR+ EVs as new biomarkers in NPC. • Provided possibility of CRISPR/Cas12a to detect trace surface proteins on EVs. [ABSTRACT FROM AUTHOR]