Clinical differentiation between Parkinson's disease (PD) and essential tremor (ET) may be challenging, highlighting the need for easily assessable diagnostic biomarkers. Neuronally derived extracellular vesicles (NDEVs) have been proposed as a peripheral matrix that can well recapitulate the cellular composition of neurons. We investigated the clinical usefulness of NDEV oligomeric and p129-α-synuclein levels in discriminating between patients with PD and those with ET. NDEV oligomeric and p129-α-synuclein species were assessed using an ELISA in 43 patients with PD, 21 patients with ET, and 45 healthy controls (HCs). NDEV oligomeric α-synuclein levels were significantly higher in PD in comparison with ET and HCs, while p129-α-synuclein values were significantly lower in HCs compared to other groups. By using a receiver operator characteristic (ROC) analysis, oligomeric-α-synuclein achieved an excellent classification performance in distinguishing PD from both ET and HCs (AUC: 0.976 and 0.997, respectively), while lower performance was obtained in differentiating ET from HCs (AUC: 0.85). On the other hand, p129-α-synuclein accurately discriminated both PD and ET from HCs (AUC: 0.997 and 0.952, respectively) but had very low performance in differentiating PD from ET (AUC: 0.47). Our study suggests that NDEV oligomeric α-synuclein is an accurate blood-derived biomarker to differentiate PD from ET, while p129-α-synuclein may be useful in distinguishing ET from HCs.