Due to a lack of early diagnostic markers, pancreatic cancer (PC) remains a lethal disease. Proteomic approaches are now being applied to identify novel PC biomarkers. In this study, iTRAQ and LC-MS/MS are used to perform comparative analyses of serum from PC patients and healthy controls (HC), to identify specific serum biomarkers for PC. Serum levels of candidate proteins are determined using ELISA. Among 869 proteins identified, 55 are potential biomarkers; Vitamin K-dependent protein Z (PROZ) and tumor necrosis factor receptor superfamily member 6b (TNFRSF6B) are selected for further analysis. Serum levels of PROZ and TNFRSF6B are significantly higher in PC patients than in HC or pancreatic benign controls (BC) (p < 0.01). The AUCs range from 0.816 to 0.971 for PROZ, TNFRSF6B, and carbohydrate antigen 19-9, either individually or in combination, in PC versus HC+BC, and from 0.711 to 0.932 in PC Stage I versus HC+BC. It is demonstrated that PROZ and TNFRSF6B are novel serum biomarkers for detecting early stage PC, and for distinguishing PC from pancreatic benign tumor and healthy individuals. Additional large cohort studies are needed to develop PROZ and TNFRSF6B as clinical PC biomarkers. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.