Objective To study the application value of serum pepsinogen (PGⅠ), carbohydrate antigen 242 (CA242), carbohydrate antigen 199 (CA199), and carcinoembryonic antigen (CEA) in the diagnosis of gastric cancer. Methods: 116 patients with gastric cancer who visited our hospital from January 2023 to January 2025 were selected as the gastric cancer group. Meanwhile, 116 healthy individuals who visited our hospital during the same period were selected as the healthy group. The levels of serum PGⅠ, CA242, CA199 and CEA in the two groups were detected and compared. Gastroscopy biopsy is taken as the "gold standard" for diagnosing gastric cancer. The diagnostic results and efficacy of PGⅠ, CA242, CA199 and CEA alone and in combination in the diagnosis of gastric cancer were statistically analyzed, and the predictive values were analyzed. Results: Compared with the healthy group, the serum PGⅠ in the gastric cancer group was significantly decreased, and the levels of CA242, CA199 and CEA were significantly increased (P < 0.05). Among 232 patients with gastric cancer and healthy individuals undergoing physical examinations, 116 positive cases and 116 negative cases were diagnosed by gastroscopy biopsy respectively. The number of true positive cases and true negative cases of PGⅠ detected was 75 and 107 respectively. The number of true positive cases and true negative cases detected by CA242 was 43 and 109 respectively. The number of true positive cases and true negative cases detected by CA199 was 46 and 104 respectively. The number of true positive cases and true negative cases detected by CEA was 41 and 105 respectively. The number of true positive cases and true negative cases detected by the combined detection of serum PGⅠ, CA242, CA199 and CEA levels was 101 and 108 respectively. The sensitivity and accuracy of the combined detection of serum PGⅠ, CA242, CA199 and CEA levels for gastric cancer were significantly improved compared with the separate detection of PGⅠ, CA242, CA199 and CEA. The missed diagnosis rate of gastric cancer in the combined detection of serum PGⅠ, CA242, CA199 and CEA levels was significantly lower than that in the separate detection of PGⅠ, CA242, CA199 and CEA (P < 0.05). The negative predictive value of combined detection of serum PGⅠ, CA242, CA199 and CEA levels for gastric cancer was significantly higher than that of separate detection of PGⅠ, CA242, CA199 and CEA (P < 0.05). Conclusion: Serum PGⅠ, CA242, CA199 and CEA are abnormally expressed in patients with gastric cancer. Combined detection can improve the accuracy and sensitivity of gastric cancer diagnosis.