The challenging diagnosis and poor prognosis of cholangiocarcinoma require the determination of biomarkers. Autoantibodies could be used in the clinic as diagnostic markers for the early detection of tumours. By proteomic approaches, several autoantibodies were proposed as potential markers. We tried in this study, to perform a serological proteome analysis, using various antigenic substrates, including tumours and human liver. Sera from patients (n = 13) and healthy donors (n = 10) were probed on immunoblots performed using 2-dimensionally separated proteins from cholangiocarcinoma cell lines (CCLP1 and CCSW1), from the liver of healthy subject and interestingly, from tumour and adjacent non-tumour liver tissues from five patients with cholangiocarcinoma and tested with their corresponding serum. Spots of interest were identified using mass spectrometry and classified according gene ontology analysis. A comparison of the whole immunoblotting patterns given by cholangiocarcinoma sera against those obtained with normal control sera enabled the definition of 862 spots. Forty-five different proteins were further analysed, corresponding to (1) spots stained with more than four of 13 (30 %) sera tested with the CCLP1 or the CCSW1 cell line and with the normal liver, and (2) to spots immunoreactive with at least two of the five sera probed with their tumour and non-tumour counter-part of cholangiocarcinoma. Immunoreactive proteins with catalytic activity as molecular function were detected at rates of 93 and 64 % in liver from healthy subjects or cholangiocarcinoma non-tumour tissues respectively, compared to 43, 33, 33 % in tumour tissues, or CCSW1 and CCLP1 cell lines. A second pattern was represented by structural proteins with rates of 7 and 7 % in normal liver or non-tumour tissues compared to 14, 33 and 67 % in tumour tissue, CCSW1 or CCLP1 cell lines. Proteins with a binding function were detected at rates of 7 % in non-tumour tissue and 14 % in tumour tissue. Using the extracted tumour tissue, serotransferrin was targeted by all cholangiocarcinoma-related sera. Immunological patterns depended on the type of antigen substrate used; i.e. tumour versus non tumour specimens. Nevertheless, a combination of multiple autoantibodies tested with the most appropriate substrate might be more sensitive and specific for the diagnosis of cholangiocarcinoma.