A novel circulating tumor-associated autoantibody, K94, obtained from a hepatocellular carcinoma (HCC) mouse model was characterized. The target antigen of K94 autoantibody was expressed in various tumor cell lines including liver cancer, and its secretion was detectable using MCF-7 breast carcinoma cells. Proteomic analysis revealed that the protein bands reactive to K94 included cytokeratin (CK) 8 and 18, which are known to be related to tumorigenesis and form a heterotypic complex with each other. However, K94 showed no activity toward CK8 or CK18 separately. The epitope of the K94 antibody was only presented by a complex between CK8 and CK18, which was confirmed by analysis using recombinant CK8 and CK18 proteins. To formulate an assay for anti-CK8/18 complex autoantibody, a mimotope peptide reactive to K94 was selected from loop-constrained heptapeptide (-CX7C-) display phage library, of which sequence was CISPDAHSC (K94p1). A mimotope enzyme-linked immunosorbent assay (ELISA) using phage-displayed K94p1 peptide as a coating antigen was able to discriminate breast cancer (n=30) patients from normal subjects (n=30) with a sensitivity of 50% and a specificity of 82.61%. CA15.3 was detected at very low levels in the same breast cancer subjects and did not discriminate breast cancer patients from normal subjects, although it is a conventional biomarker of breast cancer. These results suggest that a mimotope ELISA composed of K94p1 peptide may be useful for the diagnosis of breast cancer.