Neuron-derived extracellular vesicles (NDEVs) are potential biomarkers of neurological diseases although their reliable molecular target is not well established. Here, we demonstrate that ATPase Na ⁺ /K ⁺ transporting subunit alpha 3 (ATP1A3) is abundantly expressed in extracellular vesicles (EVs) isolated from induced human neuron, brain, cerebrospinal fluid, and plasma in comparison with the presumed NDEV markers NCAM1 and L1CAM by using super-resolution microscopy and biochemical assessments. Proteomic analysis of immunoprecipitated ATP1A3 ⁺ brain-derived EVs shows higher enrichment of synaptic markers and cargo proteins relevant to Alzheimer’s disease (AD) compared to NCAM1 ⁺ or LICAM ⁺ EVs. Single particle analysis shows the elevated amyloid-β positivity in ATP1A3 ⁺ EVs from AD plasma, providing better diagnostic prediction of AD over other plasma biomarkers. Thus, ATP1A3 is a reliable target to isolate NDEV from biofluids for diagnostic research.