Gastric cancer (GC) is a common type of cancer, particularly in China. Numerous studies have demonstrated that circulating microRNAs (miRNAs) have potential applications as noninvasive biomarkers for cancer diagnosis and prognosis. Microarray-based serum miRNA profiling was performed on the serum of 12 paired pre- and post-operative GC patients to screen differentially expressed serum miRNAs. Twelve different serum miRNAs between pre- and post-operative GC patients were identified. Those miRNAs were verified by real-time quantitative polymerase chain reaction in 110 paired pre- and post-operative serum samples from 55 GC patients. miR-20a was confirmed and demonstrated potential as a GC-associated biomarker. Furthermore, the levels of serum miR-20a were significantly different between GC, nasopharyngeal cancer, colorectal carcinoma, breast cancer and non-cancerous controls. In addition, it was found that serum miR-20a levels correlated with age, tumor stage, differentiated degree and lymph node metastasis in GC. Survival analysis indicated that GC patients with elevated levels of serum miR-20a had poor survival. Thus, serum miR-20a may serve as a molecular marker for diagnosis, evaluating therapeutic efficacy and prognosis, as well as monitoring recurrence in GC patients.