Cancer is a malignant tumor with the highest mortality of human diseases. The early diagnosis of cancer can greatly reduce its mortality. Ultracentrifugation is the most commonly employed technique to separate small extracellular vesicles (sEVs) due to their small size and rare abundance, but the low separation efficiency is a major concern. Herein, we proposed a DNAzyme-triggered assembly and disassembly system that converted single nano-sized sEVs into clusters that could be conveniently enriched by ordinary centrifugation and then be broken into single sEVs in the presence of magnesium ions. The simultaneous quantification of sEVs was realized by recording the increase in fluorescence upon nucleic acid cleavage, and a detection limit as low as 54 particles/μL was achieved. The whole analytical procedure could be completed in 1.5 h without the assistance of ultracentrifugation. Efficient enrichment and accurate quantification of sEVs are enabled through the proposed approach, broadening the potentials of sEVs in biological science, biomedical engineering, and personalized medicine.