So far, stomach-specific biomarkers, gastric cancer(GC)-related environmental factors, and cancer-associated biomarkers are three major classes of serological biomarkers with GC warning potential, joint detection of which is expected to increase the diagnosis efficiency. We investigated whether the combination of serum pepsinogens(PGs), IgG anti-Helicobacter pylori (HpAb), and osteopontin (OPN) can be used as a panel for GC diagnose. Serum was collected from 365 GC patients and 729 healthy individuals,furtherly 332 cases and 332 age- and sex-matched controls were selected for the matched analysis. Serum levels were measured by ELISA. Logistic regression and receiver operator characteristic curve (ROC) were used to assess the associations of biomarkers with GC and the discriminative performance of biomarkers for GC. The area under ROC from three-dimensional combination of PGI/II-HpAb-OPN (0.826) was significantly higher than two-dimensional combination of PGI/II-HpAb (0.786, P < 0.001), PGI/II-OPN (0.787, P < 0.001), and OPN-HpAb (0.801, P = 0.006), as well as one-biomarker of PGI/II (0.735, P < 0.001), HpAb (0.737, P < 0.001) and OPN(0.713, P < 0.001), respectively. The combination of PGI/II-HpAb-OPN, yielded a sensitivity of 70.2% and specificity of 78.3% at the predicted probability of 0.493 as the optimal cutoff point. Three-dimensional combined biomarkers assay could improve diagnostic accuracy for gastric cancer.