Objective: To investigate the application of serum miR-27b in diagnosis and prognosis of patients with osteosarcoma. Methods: Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) technique, we assessed expression of studied miR-27b in serum samples of 51 pairs of osteosarcoma patients and corresponding healthy individuals. Results: Serum levels of miR-27b were found to be significantly higher in osteosarcoma patients than those in healthy controls (P<0.001). Moreover, elevated serum levels of miR-27b in osteosarcoma patients were correlated with the large tumor size (P=0.008), advanced TNM stages (P=0.001) and distant metastasis (P=0.001). Receiver operating characteristic (ROC) curve analyses suggested that serum miR-27b had significant diagnostic value for osteosarcoma patients. It yielded an area under the curve (AUC) of ROC of 0.863 with 77.8% sensitivity and 86.7% specificity in distinguishing osteosarcoma patients from control subjects. Furthermore, Kaplan-Meier curve analysis indicated that osteosarcoma patients with a high serum miR-27b level had a significantly shortened overall survival (P<0.001). In addition, univariate and multivariable Cox regression analysis revealed that serum miR-27b level was an independent risk factor for overall survival (P<0.001 and P=0.006, respectively). Conclusion: Therefore, serum levels of miR-27b may serve as a potential non-invasive biomarker for diagnosis and prognosis of osteosarcoma patients.