Objective: Gastric cancer (GC) is a highly prevalent malignancy, yet its early diagnosis rate is generally low. Therefore, we have established a serum-based combined detection method based on tumor autoantibodies aimed at improving the diagnostic rate of gastric cancer. Methods: Through clinical studies, we selected a series of proteins aberrantly expressed in gastric cancer patients, including RalA, Survivin, NY-ESO-1, p53, Cyclin B1, and Koc, and expressed and purified them using prokaryotic expression and nickel column chromatography. Results: The levels of autoantibodies in the serum of gastric cancer patients and healthy individuals were measured using enzyme-linked immunosorbent assay (ELISA), and the diagnostic value of the combined detection of tumor autoantibodies for gastric cancer was evaluated through receiver operating characteristic (ROC) curve analysis. The levels of autoantibodies against RalA, Survivin, NY-ESO-1, p53, and Cyclin B1 in the serum of gastric cancer patients were significantly higher than those in healthy individuals (P < 0.05), while the level of Koc showed no significant difference between the two groups (P > 0.05), suggestingthat Koc may not be suitable for serological diagnosis of gastric cancer. ROC analysis of the combined levels of autoantibodies against RalA, Survivin, NY-ESO-1, p53, and Cyclin B1 for gastric cancer diagnosis achieved a sensitivity of 73.68% and specificity of 78.13%, with an AUC value of 0.8767. Conclusion: The combined tumor autoantibody detection established in this study may have promising potential applications in early screening and diagnosis of gastric cancer.