Blood Circulating miRNAs are proposed as promising biomarkers for many diseases, including Parkinson’s disease (PD). In this study, plasma circulating miRNAs expression were assessed in three independent sets with totally 151 PD, 21 Multiple system atrophy (MSA) and 138 healthy controls using high-throughput real-time PCR. 485 plasma miRNAs expression were assessed in an initial screening set of 78 PD and 78 normal controls and 7 most-differentially expressed miRNAs were selected as potential biomarkers. Following duplication test with 27 PD and 15 controls was to evaluate the performance of 7 candidate miRNAs and found 3 miRNAs (miR-320a, miR-133b, miR-221-3p) discriminate PD from controls with 74.1% sensitivity and 86.7% specificity. Moreover, miR-221-3p and miR-205 predict early PD from control and miR-205 negatively correlated with disease progression. In the third test, 4 identified miRNAs (miR-320a, miR-133b, miR-221-3p, miR-205) were evaluated in a new cohort with 46 PD, 21MSA and 45 healthy controls. As expected, the elevated miR-133b and miR-221-3p were validated to distinguish PD from controls with 82.6% sensitivity and 88.9% specificity. There was no significant difference on miR-221-3p expression between MSA and controls. Combination of up-regulated miR-133b and miR-221 has potential to serve as a noninvasive biomarker for PD diagnosis.