Objectives To evaluate the expression levels of serum microRNA-21 (miRNA-21) and microRNA-155 (miRNA-155) from patients and rats with pancreatic cancer, and to explore its value in the diagnosis of pancreatic cancer. Methods The clinical materials and the serum samples from 18 patients with pancreatic cancer (pancreatic cancer group) and 12 patients with benign pancreatic disease (benign pancreatic disease group) admitted to Fujian Medical University Union Hospital between January 2016 and December 2016 were collected prospectively. The real-time fluorescent quantitative PCR was performed to detect the levels of serum miRNA-21 and miRNA-155. 7, 12-dimethylbenz (a) anthracene (DMBA)-induced pancreatic cancer rat models (n=20) and the models of the blank control group (sham operation, n=10) were established and the serum samples from the pancreatic cancer group and the blank control group were measured by the real-time fluorescent quantitative PCR, to detect the levels of miRNA-21 and miRNA-155. Results The median expression levels of serum miRNA-21 and miRNA-155 were 1.99 (1.43–5.30) and 7.06 (4.98–21.48) in the pancreatic cancer group, as well as 1.28 (0.58–2.01) and 2.20 (1.76–3.02) in the benign pancreatic disease group. The expression levels of serum miRNA-21 and miRNA-155 were significantly higher in the pancreatic cancer group (Z=–2.621,P=0.009; Z=–3.430,P=0.001). In animal studies, the rat models of pancreatic cancer were successfully established and 11 rats with pancreatic cancer were acquired, as well as 9 rats in the blank control group were acquired. The median expression levels of serum miRNA-21 and miRNA-155 were 2.12 (1.33–2.72) and 16.45 (7.18–25.40) in the rat pancreatic cancer group, as well as 1.00 (0.45–1.60) and 1.49 (1.25–1.97) in the blank control group. The expression levels of serum miRNA-21 and miRNA-155 were significantly higher in the rat pancreatic cancer group (Z=–2.621,P=0.009; Z=–3.609,P<0.001). For distinguishing pancreatic cancer from benign diseases, the best cutoff value of serum miRNA-21 level was 4.21 and the sensitivity and specificity were 75.0% and 61.1% respectively; the best cutoff value of serum miRNA-155 level was 4.67 and the sensitivity and specificity both were 83.3%. Conclusions The serum miRNA-21and miRNA-155 levels are elevated both in patients and rats with pancreatic cancer. Detection of serum miRNA-155 will be helpful to some extent to distinguish pancreatic cancer from benign diseases.