Cholangiocarcinoma (CCA) affects the intra- and extra hepatic bile ducts and is commonly burdened by a late presentation and resulting high mortality rate. Accordingly, finding non-invasive biomarkers with adequate diagnostic/prognostic values is a priority in high-risk populations. In this study, we analyzed proteomes of serum samples from six CCA cases and ten healthy subjects using two-dimensional polyacryla mide gel electrophoresis to identify CCA-associated spots. Thirty-six CCA-associated proteins found in sera were identified by mass spectrometry. α1β-Glycoprotein (A1BG) and afamin (AFM) were detected consistently at different degrees in CCA sera compared with controls and were validated for their diagnostic and prognostic potential in a larger cohort of 64 patients with CCA, 4 with benign biliary diseases and 20 healthy subjects and compared between pre- and postsurgery serum samples from 26 CCA patients to ascertain a prognostic correlation. A single blot test developed to assess the serum A1BG/AFM ratio could detect CCA cases with 87.5% specificity, 84.4% sensitivity and the levels were significantly higher in CCA compared with controls. A high level of postoperative serum A1BG/AFM ratio was associated with worse outcomes and the infiltration of resection margins. The A1BG/AFM ratio may constitute a novel non-invasive candidate marker to diagnose CCA and its outcomes with high specificity and sensitivity. Prospective studies are awaited to demonstrate the clinical value of this observation. © 2012 by the Society for Experimental Biology and Medicine.