Altered microRNA (miRNA) associated with gastric cancer (GC) development and miR-17 and miR-106b were differentially expressed in GC tissues. This study detected serum levels of miR-17 and miR-106b expression in GC, benign gastric disease (BGD) and healthy controls to assess them as tumor markers for GC. Serum samples from 40 GC, 32 BGD (10 gastric ulcer, 14 gastric polyps, and 8 gastric ulcer with polyps) and 36 healthy individuals were subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of miR-17 and miR-106b expression. The data showed that the serum levels of miR-17 and miR-106b were significantly reduced in healthy individuals and BGD patients compared to GC patients. There was a significant association of miR-17 and miR-106b expression with age, but not with other clinicopathological features, such as gender, tumor differentiation, stage and lymphatic metastasis. Further analysis showed that, in discriminating GC patients from healthy controls, miR-17 could yield a receiver-operating characteristic (ROC) area under the curve (AUC) of 0.879 with 80.6% sensitivity and 87.5% specificity and miR-106b could yield an AUC of 0.856 with 75.0% sensitivity and 92.5% specificity. The combined AUC of miR-17 and miR-106b was 0.913 with 83.3% sensitivity and 87.5% specificity. Collectively, these data suggest that detection of serum miR-17 and miR-106b levels should be further evaluated as novel non-invasive biomarkers in early GC detection and surveillance of disease progression.