Small extracellular vesicles (sEVs) have been reported to play important roles in cell-to-cell communication and are promising biomarkers for the early diagnosis of infections. Therefore, it is in high demand to develop a method that can integrate easy-to-operate sEV isolation and sensitive quantification. We herein propose a novel detection scaffold for sEV isolation via low-speed centrifugation and the quantification of sEVs through DNAzyme-based signal amplification. The detection scaffold is established through dumbbell probe-based RCA (rolling circle amplification), containing repeated CD63 aptamer sections and DNAzyme sections. The original state of the DNAzyme section is locked in a hairpin structure in the detection scaffold. In the presence of sEVs, the CD63 aptamer recognizes and binds with sEVs, leading to the aggregation of sEVs, which can be isolated by low-speed centrifugation and the exposure of the DNAzyme section. After the catalytic fluorescence signal generation from the DNAzyme-based molecular beacon (MB) cleavage, the method exhibited a detection range of 102 to 106 particles per μL. Considering the high sensitivity and wash-free and easy-to-operate features, the strategy reported herein paves a new avenue for the effective determination of sEVs and other membrane biomolecules in fundamental and applied research.