Objective:To correlate the expression of microRNA (miRNA)-21 and miRNA-335-5p with pepsinogen and gastrin-17 in patients with gastric cancer.Methods:Sixty-one patients with gastric cancer who received treatment in Linhai Second People's Hospital between January 2019 and January 2021 were included in the patient group. An additional 60 healthy patients who concurrently received physical examination were included in the control group. Serum pepsinogen I, pepsinogen II and gastrin-17 levels were determined by latex-enhanced immunoturbidimetry. miRNA-21 and miRNA-335-5p expression were determined by quantitative reverse transcription-polymerase chain reaction. Serum pepsinogen I, pepsinogen II and gastrin-17 levels and miRNA-21 and miRNA-335-5p expression were compared between the two groups. The sensitivity and specificity of miRNA-21 and miRNA-335-5p in the diagnosis of gastric cancer were analyzed. The correlation between miRNA-21 and miRNA-335-5p expression and pepsinogen I, pepsinogen II and gastrin-17 levels was analyzed.Results:Serum pepsinogen I and gastrin-17 levels in the patient group were (54.36 ± 9.89) μg/L and (13.74 ± 1.89) pg/mL, respectively, which were significantly lower than those in the control group [(112.31 ± 23.24) μg/L, (18.75 ± 2.36) pg/mL, t = 17.89, 12.90, both P < 0.05]. Serum pepsinogen II level in the patient group was significantly higher than that in the control group [(24.35 ± 4.53) μg/L vs. (20.37 ± 3.28) μg/L, t = 5.52, P < 0.05]. The relative mRNA expression of miRNA-21 in the patient group was significantly higher than that in the control group [(3.42 ± 0.61) vs. (0.53 ± 0.12), t = 30.01, P < 0.05]. The relative mRNA expression of miRNA-335-5p in the patient group was significantly lower than that in the control group [(0.32 ± 0.17) vs. (1.65 ± 0.35), t = 26.65, P < 0.05]. The receiver operating characteristic curve analysis showed that the sensitivity and specificity of miRNA-21 in the diagnosis of gastric cancer were 74.36% and 68.18%, respectively, and they were 79.49% and 60.90% for miRNA-335-5p, respectively. There was a negative linear correlation between miRNA-21 and pepsinogen I and gastrin-17 levels ( r = -0.82, -0.74), but there was a positive linear correlation between miRNA-21 and pepsinogen II levels ( r = 0.76). There was a positive linear correlation between miRNA-335-5p and pepsinogen I and gastrin-17 ( r = 0.79, 0.72), but there was a negative linear correlation between miRNA-335-5p and pepsinogen II levels ( r = -0.70). Conclusion:miRNA-21 is highly expressed in patients with gastric cancer, while miRNA-335-5p is lowly expressed. miRNA-21 and miRNA-335-5p are highly correlated with pepsinogen and gastrin-17 levels. miRNA-21 and miRNA-335-5p can be used as effective indices for diagnosis of gastric cancer. Findings of this study are highly innovative and scientific.