Extracellular vesicle-associated miRNAs (EV-miRNAs) are emerging as a new type of noninvasive biomarker for disease diagnosis. Their relatively low abundance, however, makes accurate detection challenging. Here, we designed a DNA nanowire guided-catalyzed hairpin assembly (NgCHA) nanoprobe for profiling EV-miRNAs. NgCHA showed high penetrability to EVs, which allowed rapid delivery of the probes into EVs. In the presence of targeted miRNAs within EVs, a fluorescent signal could be generated and amplified by confining the catalytic hairpin assembly system within the nanowires, thus greatly enhancing the analytical sensitivity. We showed that EV-miRNAs from various cell lines could be accurately quantified by NgCHA in situ. By using a four-EV-miRNA panel, this platform can identify patients with breast cancer at an early stage with 95.2% sensitivity and 86.7% specificity. Its applications for risk assessment as well as cancer type prediction were also successfully demonstrated. This platform is sensitive, low-cost, and simple compared with current methods. It may thus serve as a promising tool for the noninvasive diagnosis and monitoring of cancers and other diseases through EV-miRNA profiling.