Extracellular vesicles (EVs) derived from diverse biofluids have emerged as promising noninvasive diagnostic biomarkers or signatures for various diseases. EV-associated N-glycoproteins are crucial for the biological functions of EVs and hold significant potential for disease diagnosis and prognosis. However, analyzing changes in EV N-glycoproteins from ultratrace samples presents considerable challenges. This study introduces a robust method, which utilizes magnetic nanochains to sensitively enrich EV N-glycoproteins in a single tube.