Objectives: Pancreatic cancer is one of the most common malignant tumors of the digestive tract. Due to its strong concealment and rapid disease progress, it is characterized by high mortality and low cure rate. Current research focuses on screening high-risk populations, preventing the occurrence of pancreatic cancer and developing imaging technology and tumor markers for early diagnosis. This study aimed to investigate the absolute quantitative detection of microRNA-25 (miR-25) and reveal its quantitative changes in the treatment of pancreatic cancer. We compared serum miR-25 level between pancreatic cancer patients and other tumor patients, especially those with gastrointestinal cancer, to provide further evidence for early diagnosis, differential diagnosis and prognosis of pancreatic cancer. Methods: We collected serum samples from 51 pancreatic cancer patients (including 21 patients with preoperative and postoperative samples), 52 pancreatitis patients, 141 other tumor patients, and 50 healthy individuals from Huashan Hospital, Fudan University. The serum levels of miR-25, Carbohydrate Antigen 19–9 (CA19–9), Carbohydrate Antigen 125 (CA125) and Carcinoembryonic Antigen (CEA) in these populations were measured to analyze the value of miR-25 quantitative detection in the diagnosis, postoperative assessment and Tumor Node Metastasis (TNM) staging of pancreatic cancer. Results: Among the 51 pancreatic cancer patients, 50 cases (98.04 %) showed miR-25 levels above the threshold (3333 copies/μl), while all samples in normal group showed negative. The mean level of miR-25 in serum was 5707.45 ± 361.02 copies/μl. In other tumor groups, 21/141 (14.89 %) patients showed positive results and the mean miR-25 level was 847.09 ± 125.97 copies/μl. Comparing before and after operation in 21 paired samples, the level of miR-25 declined significantly after operation, with an average decline rate of 86.36 %. Conclusions: Serum miR-25 levels were significantly higher in pancreatic cancer patients compared to both normal and other tumor groups and decreased significantly after surgery. In addition, miR-25 showed higher sensitivity than common tumor markers (CA19–9, CA125, CEA) in early diagnosis of pancreatic cancer, and proved to be of significant value in evaluating the effect of surgical treatment.