Objective To explore the expression of serine/threonine protein kinase 1 （AKT1） and Isochorismatase Domain-Containing 1 （ISOC1） in gastric cancer tissues and evaluate their diagnostic utility. Methods A total of 90 patients with gastric cancer who were diagnosed and treated in our hospital between March 2023 and September 2024 were recruited as the study subjects. The positive expression rates and positive scores of AKT1 and ISOC1 in cancer tissues and adjacent tissues were compared. The AKT1 and ISOC1 positive scores of gastric cancer patients with diverse characteristics were also compared. The Spearman correlation analysis was employed to examine the relationship between the expression of AKT1 and ISOC1 in cancer tissues and the clinical features of gastric cancer patients. A diagnostic model for gastric cancer， integrating AKT1 and ISOC1， was established using the Logistic regression model. Receiver operating characteristic （ROC） curves were plotted to analyze the area under the curve （AUC） value， sensitivity， and specificity of AKT1， ISOC1， and their combined diagnosis for gastric cancer. Results The positive expression rates and positive scores of AKT1 and ISOC1 in cancer tissues were significantly higher than those in paracancerous tissues （P < 0.05）. In gastric cancer patients， the expressions of AKT1 and ISOC1 were notably higher in patients with low differentiation， clinical stage Ⅰ~Ⅱ， invasion depth T_1-2， absence of lymph node metastasis， and no distant metastasis compared to those with moderate differentiation， clinical stage Ⅲ~Ⅳ， invasion depth T_3-4， and presence of lymph node metastasis and distant metastasis （P < 0.05）. Spearman's correlation analysis indicated that AKT1 and ISOC1 were positively correlated with the degree of differentiation， clinical stage， lymph node metastasis， invasion degree， and distant metastasis （P < 0.05）. The area under the curve （AUC） values of AKT1， ISOC1， and their combined diagnosis for gastric cancer were 0.735， 0.726， and 0.875 respectively （P < 0.05）. The sensitivities were 60.00%， 56.70%， and 75.60%， while the specificities were 85.60%， 83.30%， and 87.80%. The AUC value of the combined detection of AKT1 and ISOC1 in the diagnosis of gastric cancer was higher than that of AKT1 or ISOC1 alone （Z = -3.003， -3.196， P < 0.05）. Conclusions AKT1 and ISOC1 are highly expressed in gastric cancer tissues， and their expressions are upregulated with the progression of the disease. The combined detection of their expression levels holds great significance for the diagnosis and prognosis assessment of gastric cancer.