MicroRNAs are potentially very useful biomarkers in the diagnosis of cancer. We sought to identify specific microRNAs in peripheral blood mononuclear cells (PBMCs) whose levels might facilitate diagnosis of pancreatic cancer. We investigated PBMC microRNA expression in three independent cohorts [healthy, benign pancreatic/peripancreatic diseases (BPD), and pancreatic cancer], comprising a total of 352 participants. First, we used sequencing technology to identify differentially expressed microRNAs in PBMC of pancreatic cancer, BPD, and healthy controls (n = 20 in each group). Then the selected microRNAs were analyzed using the quantitative reverse transcriptase PCR assays in the remaining 292 samples. The predictive value of the microRNAs was evaluated by logistic regression models and the receiver operating characteristic curve (AUC). We found that miR-27a-3p level in PBMCs could discriminate pancreatic cancer from BPD with a sensitivity of 82.2% and specificity of 76.7% (AUC = 0.840; 95% CI, 0.787-0.885%). Combination of PBMC miR-27a-3p and serum CA19-9 levels provided a higher diagnostic accuracy with a sensitivity of 85.3% and specificity of 81.6% (AUC = 0.886; 95% CI, 0.837-0.923%). The satisfactory diagnostic performance of the panel persisted regardless of disease status (AUCs for tumor-node-metastasis stages I-III were 0.881, 0.884, and 0.893, respectively). PBMC miR-27a-3p level represents a potential marker for pancreatic cancer screening. A panel combining serum CA19-9 and PBMC miR-27a-3p level could have considerable clinical value in diagnosing pancreatic cancer.