BACKGROUND: Pathological α-synuclein aggregates are key finding of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. The Real-Time Quaking-Induced Conversion (RT-QuIC) assay using cerebrospinal fluid (CSF) can sensitively detect pathological α-syn aggregates with strong biologic rationale. Because of limitation by the invasive nature of CSF collection, blood-based RT-QuIC assay is now attended with small evidence and showed good performance discriminating PD and control. This study investigated pathological α-syn aggregates using the neuronal extracellular vesicles (nEVs) from serum in patients with synucleinopathies, and healthy controls (HC). METHODS: Serum were collected from the patients diagnosed with synucleinopathies and HC without neurological disorders. Total extracellular vesicles (tEVs) were isolated from serum using ExoQuick kit, followed by nEVs isolation via L1-Cell Adhesion Molecule (L1CAM) immunocapture. The identity of nEVs was confirmed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. Pathological α-synuclein aggregates in EVs were assessed by western blotting, dot blot, and RT-QuIC assay. RESULTS: The concentration and abundance of EVs were comparable between PD and HC groups. TEM and NTA confirmed extracellular vesicle morphology, and size distribution, and western blotting validated neuronal quality of nEVs. Higher levels of phosphorylated or aggregated α-synuclein were detected in nEVs from synucleinopathy patients compared to HC. Optimized RT-QuIC conditions achieved 100% sensitivity without false positives. Distinct RT-QuIC kinetic profiles were observed between synucleinopathy and HC samples. CONCLUSION: Serum-derived nEVs represent a promising, minimally invasive seed source for RT-QuIC assays, offering robust diagnostic performance for the detection of synucleinopathy and potential applicability in broader clinical settings.