Cancer-cell-derived exosomes are regarded as noninvasive biomarkers for early cancer diagnosis because of their critical roles in intercellular communication and molecular exchange. A robust aptamer-initiated catalytic hairpin assembly (AICHA) fluorescence assay is proposed for universal, sensitive detection of cancer-derived exosomes. The AICHA was verified with the specific detection of MCF-7 cell-derived exosomes with a wide calibration range of 8.4 particles/μL to 8.4 × 105 particles/μL and a low detection limit (LOD) of 0.5 particles/μL. The universality of the AICHA method was verified for PANC-1 cell-derived exosomes, the LOD of which was determined to be 0.1 particles/μL. The performances in serum samples were detected with a recovery rate range of 95.45-106.2%, which demonstrates its significant potential for protein biomarker analysis and cancer diagnosis.