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Previously, we identified six miRNAs that are differentially expressed in colorectal cancer compared with healthy controls. Here, we tested them in gastric cancer GC.We performed quantitative RT-PCR on serum samples from 92 patients with gastric cancer and 89 controls for the six miRNAs, and analyzed their risk scores to evaluate the diagnostic value of the serum miRNA profiling system.After a two-phase selection and validation process, five miRNAs were found to significantly differ in expression between gastric cancer samples and control samples, including miR-21, miR-31, miR-92a, miR-181b, and miR-203. Risk score analysis showed that this miRNA panel could distinguish gastric cancer cases from controls with high sensitivity and specificity. Under receiver operating characteristic curves, areas under the curve for tumor identification were 0.933 (95% confidence interval [CI]: 0.86–1.007) for the training set and 0.919 (95% CI: 0.863–0.975) for the validation set—markedly higher than those of carcinoembryonic antigen (0.624) and carbohydrate antigen 19-9 (0.603).The signature of these five miRNAs is a novel and noninvasive biomarker for gastric cancer, and could facilitate and simplify its diagnosis.