Extracellular vesicles (EVs) have emerged as promising tumor biomarkers for early cancer diagnosis, as primary tumor-secreted EVs carry characteristic molecular information on parent cells. It is thus desirable to realize the efficient discrimination of the signatured EVs-associated microRNAs (miRNAs) with low expression and subtle variation. Here, we introduce an autonomous nonlinear enzyme-free signal amplification paradigm for EVs discrimination through a highly sensitive and selective detection of their inherent miRNAs in situ. Our proposed amplifier consists of a modularized DNAzyme-amplified two-stage cascaded hybridization chain reaction (CHCR–DNAzyme) circuit, where the analyte-generated output of the preceding hybridization chain reaction (HCR1) stage serves as input to motivate the following hybridization chain reaction (HCR2) stage and the concomitant assembly of numerous DNAzyme biocatalysts. By incorporating a flexibly configurable sensing module, this modular CHCR–DNAzyme circuit can further extend to “plug-and-play” sensing mode that enables the miRNA assay with high specificity. The sophisticated design and the detecting performance of our CHCR–DNAzyme scheme were systematically investigated in vitro. The optimized CHCR–DNAzyme system was further applied for distinguishing EVs derived from different cells through the amplified detection of a putative miRNA biomarker in EVs. This compact CHCR–DNAzyme amplifier provides a universal and facile toolbox for highly efficient identification of multiple miRNAs-involved EVs and thus holds great potential for early cancer diagnosis.