Autoantibodies, which are generated by immune system recognizing the presence of the abnormal tumor-associated antigens, are promising biomarkers for early detection of tumors. Recently, we established a B cell hybridoma pool derived from H-ras12V transgenic mouse, a typical hepatocellular carcinoma model, as a source of tumor-associated autoantibodies without using any extracellular antigens and have characterized the specific target antigens against them. K1 autoantibody, one of them, was investigated in this study and its target antigen was identified by mass spectrometric analysis as fatty acid synthase (FASN), an important oncogenic protein. Moreover, a specific mimotope against K1 autoantibody was screened from the cyclic random hepta-peptide phage library and, using it as a coating antigen for ELISA, we could distinguish patients with hepatocellular carcinoma (HCC) vs. normal subjects with 96.55% sensitivity and 100% specificity. These results imply that anti-FASN autoantibody is induced in patients with HCC and detection of anti-FASN autoantibody can be used for the diagnosis of HCC.