Objective To investigate the correlations of the expressions of microRNA-506 (miR-506) and microRNA-874 (miR-874) in serum of patients with gastric cancer with Helicobacter pylori (Hp) infection and severity of disease. Methods A total of 78 patients with gastric cancer admitted to our hospital from May 2020 to May 2022 were selected in the study, and the patients were divided into infection group (43 cases) and non-infection group (35 cases) according to Hp infection. Pearson method was applied to analyze the correlation between the expression levels of miR-506 and miR-874 in serum of patients; Logistic regression was used to analyze the related factors affecting the severity of the disease, and receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of miR-506 and miR-874 in Hp infection and severity of the disease. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of miR-506 and miR-874 in Hp infection and disease severity. Results The serum miR-506 level in the infection group was higher than that in the non-infection group, and the expression level of miR-874 was lower than that in the non-infection group (1.22±0.27 versus 1.50±0.51, P < 0.05); the serum levels of miR-506 and gastrin-17 (G-17) in patients of stage Ⅲ to Ⅳ were higher than those in patients of stage Ⅰ to Ⅱ, and the expression levels of miR-874 and pepsin Ⅰ (PG Ⅰ) were lower than those in patients of stage I to Ⅱ (P < 0.05). The expression levels of miR-506 and miR-874 in serum were negatively correlated (r=-0.886, P < 0.05). There were no correlations of age, sex, tumor diameter and differentiation degree with the severity of disease (P>0.05). The lymph node metastasis rate in patients with stage Ⅲ to Ⅳ was higher than that in patients with stage Ⅰ to Ⅱ(P < 0.05). Logistic regression analysis showed that miR-506 and miR-874 were the influencing factors of severity of the disease (P < 0.05). ROC curve analysis results showed that the area under the curve (AUC) of serum miR-506, miR-874 and their combined detection for diagnosis of Hp infection were 0.741, 0.648 and 0.823, respectively. The combined detection was better than that of serum miR-506 and miR-874 alone (P=0.007, 0.001). The AUC of serum miR-506, miR-874 and their combined detection for diagnosis of disease severity were 0.697, 0.797 and 0.836, respectively. The predictive efficiency of the combined detection of the two methods was better than that of serum miR-506 and miR-874 separately (P=0.045, 0.046). Conclusion The expression levels of miR-506 and miR-874 in serum are related to the occurrence and severity of Hp infection. The combined detection of the combined detection has certain auxiliary diagnostic value for Hp infection and severity of the disease.