Background Post-operative pancreatic fistula (POPF) is a common and potentially life-threatening complication following pancreaticoduodectomy. The aim of this study was to assess the predictive value of intra-operative amylase concentration (IOAC) in peri-pancreatic fluid after resection for the diagnosis of POPF. Methods Consecutive patients who underwent a pancreaticoduodectomy between September 2014 and October 2015 were included in the analysis. IOAC was measured intraoperatively followed by drain fluid analysis for amylase on post-operative days (POD) 1, 3 and 5. Receiver operator characteristic (ROC) analysis was performed to evaluate the discriminative capacity of IOAC as a predictor of POPF. Results IOAC was measured after pancreaticoduodectomy in 62 patients. The IOAC correlated significantly with i) POD 1 and 3 drain amylase (p < 0.01), ii) the development of POPF (p < 0.01), iii) the development of clinically relevant fistula (Type B, C) (p < 0.01), iv) delayed gastric emptying (p < 0.01), and v) grade of complication as per the Clavien-Dindo definition (p = 0.02). ROC curve analysis confirmed the predictive relationship of IOAC and POPF as a good test with an area under the curve of 0.93, 95% CI 0.87–0.99, p < 0.01. In patients with IOAC of 200 U/L or higher the POPF rate was 80% (OR = 50.1, p < 0.0001). Discussion Measurement of IOAC allows early and accurate categorization of patients at risk for POPF.