Detection of oligomeric alpha-synuclein (O-alpha-syn) in blood plasma as a potential diagnostic biomarker for Parkinson's disease (PD) has obtained inconsistent results. The abundance of alpha-syn in red blood cells (RBCs) provides a novel option for detecting the RBC-derived alpha-syn in PD diagnosis. Previous studies have shown levels of O-alpha-syn in RBCs (O-alpha-syn-RBC) are increased in patients with PD and multiple system atrophy (MSA). However, whether the levels of O-alpha-syn-RBC can reflect the change of alpha-syn in the brain and be used as a biomarker for differential diagnosis between PD and MSA remains poorly understood. In the present study, levels of O-alpha-syn RBC in participants with PD (n = 333), MSA (n = 114), and healthy controls (n = 334) were measured using an enzyme linked immunosorbent assay. The levels of O-alpha-syn-RBC were significantly higher in PD patients than in MSA patients (P < 0.0001) and healthy controls (P < 0.0001). Receiver operating characteristic curve (ROC) indicated that the O-alpha-syn-RBC level could clearly separate PD patients from healthy controls (sensitivity: 76.58%; specificity: 77.06; area under the curve (AUC): 0.82). The O-alpha-syn-RBC level could also differentiate PD from MSA patients (sensitivity: 85.29%; specificity: 61.40%; AUC: 0.78). However, no significant difference was detected in the levels of O-alpha-syn-RBC between PD and MSA patients. To investigate the correlation between O-alpha-syn-RBC levels and brain alpha-syn expressions, rat models with striatum overexpression of human alpha-syn were developed, and the effect of brain alpha-syn expression on the levels of O-alpha-syn-RBC was investigated. Overexpression of alpha-syn in one side of the striatum led to a significant increase of O-alpha-syn levels in the ipsilateral striatum, and simultaneously a dramatic elevation of O-alpha-syn-RBC levels in the periphery. Extracellular application of monomeric alpha-syn and O-alpha-syn also resulted in a rapid elevation of O-alpha-syn-RBC levels. Taken together, the present study suggests that O-alpha-syn-RBC levels are affected by brain a-syn expression and this could be used as a potential biomarker for separating PD from MSA patients and healthy controls.