Background: Multiple system atrophy(MSA) is a neurodegenerative disease characterized by intracellular a-synuclein deposits. There is an unmet need for blood-based biomarkers to diagnose MSA. Our previous studies have reported elevated alpha-synuclein levels in erythrocytes of MSA patients. However, alpha-synuclein protein in the membrane and cytoplasm of erythrocytes in MSA have not been investigated. Methods: The membrane and cytoplasm were extracted from erythrocytes in 77 patients with MSA and 133 healthy controls. Levels of total and oligomeric alpha-synuclein were detected using Electrochemiluminescence assays. The correlations between alpha-synuclein levels and clinical characteristics were explored in MSA group. The diagnostic value of erythrocyte alpha-synudein for MSA was determined by Receiver operator characteristic curve. Results: alpha-synuclein levels in the erythrocyte membrane were significantly elevated in MSA patients compared with the healthy controls (total alpha-synuclein, p = 0.003; oligomeric alpha-synuclein/total alpha-synuclein, p = 0.033; oligomeric alpha-synuclein/protein, p < 0.001). The combination of total and oligomeric alpha-synuclein levels in erythrocyte membrane could efficiently distinguish MSA from healthy controls (sensitivity of 79.2%; specificity of 69.2%; area under the curve: 0.771). In contrast, no significant difference was found in erythrocyte cytoplasm alpha-synuclein levels. In the subgroup of 48 patients with probable MSA, there was a weakly negative correlation between oligomeric alpha-synuclein/protein in erythrocyte membrane and disease duration (r = -0.336; p = 0.009). Conclusion: Our pilot study suggests that the membrane fraction of alpha-synuclein levels in erythrocyte were elevated in patients with MSA, and these levels may be decreased with the development of disease.