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BACKGROUND The current study aims to explore the clinical value of serum miR-499a-5p in the diagnosis of pancreatic cancer. METHODS One hundred twenty-four patients with pancreatic cancer (cancer group), 100 patients with benign pancreatic diseases (benign control group), and 100 healthy people (healthy control group) were selected as the observation objects from January 2017 to June 2017. Fasting venous blood samples were collected to detect the levels of CA199 and the relative expression of miR-499a-5p in serum, and to evaluate the diagnostic value for pancreatic cancer. RESULTS The expression of CA199 in the benign control group and cancer group was significantly higher than that in the healthy control group. However, the expression of miR-499a-5p in the cancer group was significantly higher than that in the benign control group and the healthy control group. But no difference of serum miR-499a-5p level was found in the benign control group and the healthy control group. Receiver operating characteristic (ROC) analysis showed that when used alone, the sensitivity and specificity of miR-499a-5p in the diagnosis of pancreatic cancer were better than that of CA199 (p < 0.05). Moreover, when serum miR-499a-5p was combined with CA199, the diagnostic value for pancreatic cancer increased significantly (p < 0.05). Dual luciferase reporter assay showed that PTEN was a target gene of miR-499a-5p. CONCLUSIONS In summary, miR-499a-5p is a new, non-invasive biomarker, and the combination of miR-499a-5p and CA199 can improve the diagnostic sensitivity of pancreatic cancer.