Lay abstract Gastric cancer is the most common cancer type in the digestive system. In this study we developed an effective and convenient blood-based test to detect gastric cancer. This test combined a conventional protein test with a newly invented methylation test. We aimed to validate the test and examine its performance using a prospective cohort study. The study showed that the test has promising potential for noninvasive early detection of gastric cancer. We found that single protein or methylation markers detected a proportion of gastric cancer cases, while a combination of these markers exhibited maximal detection capability. Therefore we concluded that the combined test provided adequate efficacy and should be used as a strategy for future gastric cancer detection. Aim: This study aimed to validate a combination of mSEPT9, mRNF180 and CA724 for gastric cancer (GC) detection. Patients & methods: The performance of mSEPT9, mRNF180 and CA724 was examined in a prospective cohort study with 518 participants (151 with GC, 56 with atrophic gastritis, 87 with other gastrointestinal diseases and 224 with no evidence of disease). Results: mSEPT9, mRNF180 or CA724 alone detected 48.3, 37.1 and 43.1% of GC, respectively. The combination of mSEPT9 and mRNF180 detected 60.3% of GC, and the combination of all three markers detected 68.6% of GC. The detection sensitivity of mSEPT9 and mRNF180 was significantly higher for gastric body and in elder subjects. mSEPT9 was correlated with poorer GC survival. Conclusion: The combination of mSEPT9, mRNF180 and CA724 was adequately sensitive for GC detection. The blood mSEPT9 was predictive for GC prognosis.