Extracellular vesicles (EVs) are small, membrane-bound particles released by all cells that have emerged as an attractive biomarker platform. We study the utility of a dielectrophoretic (DEP) micro-chip device for isolation and characterization of EVs derived from plasma specimens from patients with brain tumors. EVs were isolated by DEP chip and subjected to on-chip immunofluorescence (IF) staining to determine the concentration of glial fibrillary acidic protein (GFAP) and Tau. EVs were analyzed from the plasma samples isolated from independent patient cohorts. Glioblastoma cell lines secrete EVs enriched for GFAP and Tau. These EVs can be efficiently isolated using the DEP platform. Application of DEP to clinical plasma samples afforded discrimination of plasma derived from brain tumor patients relative to those derived from patients without history of brain cancer. Sixty-five percent (11/17) of brain tumor patients showed higher EV-GFAP than the maximum observed in controls. Ninety-four percent (16/17) of tumor patients showed higher EV-Tau than the maximum observed in controls. These discrimination thresholds were applied to plasma isolated from a second, independent cohort of 15 glioblastoma patients and 8 controls. For EV-GFAP, we observed 93% sensitivity, 38% specificity, 74% PPV, 75% NPV, and AUC of 0.65; for EV-Tau, we found 67% sensitivity, 75% specificity 83% PPV, 55% NPV, and AUC of 0.71 for glioblastoma diagnosis. This proof-of-principle study provides support for DEP-IF of plasma EVs for diagnosis of glioblastoma.