Gastric cancer (GC) is one of the most common causes of cancer deaths worldwide; however, reliable and non-invasive screening methods for GC are not established. Therefore, we conducted this study to develop a biomarker for GC detection, consisting of urinary microRNAs (miRNAs). We matched 306 participants by age and sex [153 pairs consisting of patients with GC and healthy controls (HCs)], then randomly divided them across three groups: (1) the discovery cohort (4 pairs); (2) the training cohort (95 pairs); and (3) the validation cohort (54 pairs). There were 22 urinary miRNAs with significantly aberrant expressions between the two groups in the discovery cohort. Upon multivariate analysis of the training cohort, urinary expression levels of miR-6807-5p and miR-6856-5p were significantly independent biomarkers for diagnosis of GC, in addition to Helicobacter pylori (H. pylori) status. A diagnostic panel that combined these 2 miRNAs and H. pylori status distinguished between HC and GC samples with an area under the curve (AUC) = 0.736. In the validation cohort, urinary miR-6807-5p and miR-6856-5p showed significantly higher expression levels in the GC group, and the combination biomarker panel of miR-6807-5p, miR-6856-5p, and H. pylori status also showed excellent performance (AUC = 0.885). In addition, this biomarker panel could distinguish between HC and stage I GC patients with an AUC = 0.748. Urinary expression levels of miR-6807-5p and miR-6856-5p significantly decreased to undetectable level after curative resection of GC. This novel biomarker panel enables early and non-invasive detection of GC.