The detection of α-synuclein (α-syn) in the cerebrospinal fluid (CSF) of patients with synucleinopathy has yielded promising but inconclusive results. The aim of the present study was to determine the diagnostic value of α-syn as a biological marker for Dementia with Lewy bodies (DLB) vs. normal subjects and patients with Alzheimer's disease (AD), after strict control of several recognized confounders. Sixteen patients with DLB, 18 patients with AD and 22 age- and sex-matched normal controls (CTRL) were recruited. The levels of total α-syn in CSF were measured using a novel enzyme-linked immunosorbent assay. There was a significant increase of CSF α-syn levels in DLB patients as compared to the CTRL and AD groups (P = 0.049 and 0.01 respectively). ROC analysis revealed that increased α-syn was 81.8% specific for the discrimination of DLB vs. CTRL and 90% vs. AD. However, sensitivity was lower (56.2 % and 50% respectively). These findings provide evidence for a possible diagnostic role of α-syn as a surrogate biomarker for DLB.