Extracellular vesicles (EVs) are emerging circulating biomarkers and contain diverse sugar modifications (glycans) that can mediate disease progression. The analysis of EV glycans, however, is challenging due to technology limitations. We have developed a dedicated platform to directly profile EV glycans in native biofluids. The all-magnetic technology utilizes rationally designed nanoparticles and integrated sensors to specifically measure EV-bound glycans but not that of free-floating proteins. The assay is fast, sensitive, and wash-free, and reveals new clinical signatures to differentiate patient prognosis.