Current biomarkers for diagnosis and prognosis prediction in gastric cancer (GC) and gastroesophageal junction (GEJ) cancer have limited accuracy, and the role of soluble immune checkpoints is unknown. In this study, T cell immunoglobulin and mucin domain-containing-3 (TIM-3) and galectin-9 expression was investigated in single-cell RNA (scRNA) sequencing data and with multiplex immunohistochemistry. Further, serum samples from 310 patients with GC/GEJ and 82 healthy donors were analyzed for soluble TIM-3 (sTIM-3) and galectin-9 (sGal-9). sGal-9 levels were significantly increased in patients with cancer compared to healthy donors (p < 0.001). Notably, the combination of sTIM-3 and sGal-9 had a higher diagnostic accuracy for GC/GEJ than the established tumor markers CEA, CA19-9, and CA72-4. Patients with GC/GEJ with increased sTIM-3 and sGal-9 levels had a significantly reduced overall survival with a hazard ratio of 1.77 (sTIM-3 high), 1.82 (sGal-9 high), and 2.27 (sTIM-3 high+sGal-9 high), respectively. Collectively, sTIM-3 and sGal-9 may serve as novel non-invasive biomarkers for diagnosis and outcome prediction in patients with GC/GEJ.