The present study was designed to investigate cholangiocarcinoma (CCA) antibodies in hamster serum.Hamster CCA cell lines were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis andmatrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A candidate biomarker wasconfirmed by immunoprecipitation and western blot, and was further analyzed using ELISA and sera fromnormal control hamsters, hamsters with opisthorchiasis and hamsters with various stages of CCA, as well asfrom CCA patients and healthy individuals. One candidate marker was identified as HSP90α, as indicated bya high level of anti-HSP90α in hamster CCA sera. It was found that the levels of anti-HSP90α were specificallyelevated in the sera of hamsters with CCA compared with other groups and progressively increased with theclinical stage. At the cut-off point of 0.4850 on the receiver operating characteristic curve, anti-HSP90α coulddiscriminate CCA from healthy control groups with a sensitivity of 76.2%, specificity of 71.4% and total accuracy75.5%. In the present study, we have shown that anti-HSP90α may be a potential useful serum biomarker todiscriminate CCA cases from healthy persons.