Objective. To explore the clinical value on combined detection of serum carbohydrate antigen 724 (CA724), secreted protein dickkopf-1 (DKK1), and thymidine kinase 1 (TK1) in the diagnosis of gastric cancer (GC). Methods. The clinical data of 63 GC patients (GC group) and 54 patients with benign gastric lesions (control group) admitted to Zhu Xianyi Memorial Hospital of Tianjin Medical University from June 2020 to June 2021 were retrospectively analyzed. The levels of serum CA724, DKK1, and TK1 in the two groups were detected by electrochemiluminescence instrument, enzyme-linked immunosorbent assay, and enhanced chemiluminescence. The diagnostic efficacy of single detection and combined detection was analyzed by drawing the receiver operating characteristic (ROC) curve. Results. Compared with the control group, the serological indexes of patients in GC group were markedly higher ( P < 0.001 ). ROC curve analysis showed that the areas under the curve of serum CA724, DKK1, TK1, and combined detection in the diagnosis of GC were 0.849, 0.754, 0.685, and 0.923, respectively; and the sensitivity and specificity of their combined detection were higher than those of separate detection. Conclusion. The levels of serum CA724, DKK1, and TK1 were highly expressed in GC patients, with a higher diagnostic value for GC in their combined detection, which can effectively screen and assist the diagnosis of GC.