Cholangiocarcinoma (CCA) is a rare but devastating neoplasm that accounts for about 3% of all gastrointestinal cancers and about 15% of all primary liver cancers worldwide. The lack of early detection and limited therapeutic options are major problems in controlling CCA. The current study attempted to identify novel serum markers which can substitute the carbohydrate antigen CA19-9, or can improve, when measured together, the diagnostic accuracy of CA19-9. Differentially expressed proteins in pooled and individual plasma samples obtained from patients with CCA and control subjects (10 each) were identified by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MALDI-TOF). Out of a total of 21 protein spots separated and identified, five spots were found to be up-regulated in plasma from CCA patients. The up-regulation of α1-antitrypsin (AP1) was observed in all of the ten samples from CCA patients with protein intensity significantly higher than control subjects. Based on results of binary logistic regression analysis of the three serum biomarkers (CA19-9, AP1 and α-fetoprotein: AFP), serum levels of at least CA19-9 together with AP1 were the minimum requirement to obtain prediction accuracy of greater than 80% in a battery test for diagnosis of CCA. However, in order to obtain high predictability of 100% or approaching, an addition of at least one of the three liver function enzymes (alkaline phosphatase: ALP; aspartase transaminase: AST; alanine trasaminase: ALT) is required. Serum biomarkers may be a useful diagnostic or prognostic monitoring tool for CCA. Further evaluation of larger number samples is needed to support their applicability in a clinical setting as diagnostic and prognostic tools. Determination of clinical utility of these marker models in early diagnosis of CCA requires study in animal models with disease progression.