Osteosarcoma is the most common human primary malignant bone tumor in children and young adults. Sensitive and non-invasive biomarkers that can facilitate disease detection at early stage are highly desirable to improve survival rate and help to determine optimized treatment for osteosarcoma. The small non-coding RNAs, microRNAs (miRNAs), have recently been identified as critical regulators for various diseases including cancer and may represent a novel class of cancer biomarkers. In this study, we aimed to detect the potential of circulating miRNAs as biomarkers for osteosarcoma. Levels of six candidate miRNAs (miR-21, miR-199a-3p, miR-143, miR-34, miR-140, and miR-132) that were previously demonstrated to be regulated in osteosarcoma were examined in plasma of 40 osteosarcoma patients and 40 matched healthy controls by quantitative reverse-transcription polymerase chain reaction assays. The results showed that circulating levels of miR-21 were significantly higher in osteosarcoma patients than controls, while miR-199a-3p and miR-143 were decreased in osteosarcoma patients. We replicated the findings in an independent study of 40 osteosarcoma patients and 40 matched controls and confirmed the results. Receiver operating characteristics curve analysis of the combined populations demonstrated that the three-miRNA signature could discriminate cases from controls with an area under the curve of 0.953 (95 % CI 0.924–0.984). In addition, circulating miR-21 and miR-143 were correlated with both metastasis status and histological subtype of the patients, while miR-199a-3p only correlated with histological subtype. Our data suggest that altered levels of circulating miRNAs might have great potential to serve as novel, non-invasive biomarkers for osteosarcoma.