Previous studies demonstrated that cancer sera contain antibodies, which react with a unique group of autologous cellular antigens called tumour-associated antigens (TAAs). This study determines whether a mini-array of multiple TAAs would enhance antibody detection and be a useful approach in colon cancer detection and diagnosis. The mini-array of multiple TAAs was composed of five TAAs including Imp1, p62, Koc, p53 and c-myc full-length recombinant proteins. Enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against these five TAAs in 46 sera from patients with colon cancer and also 58 sera from normal individuals. Antibody frequency to any individual TAA in colon cancer was variable and ranged from 15.2% to 23.9%. With the successive addition of TAAs to a final total of five antigens, there was a stepwise increase of positive antibody reactions reaching a sensitivity of 60.9% and a specificity of 89.7% in colon cancer. Positive and negative likelihood ratio was 5.91 and 0.43 respectively, which showed that the clinical diagnostic value of parallel assay of five TAAs was high. Positive and negative predictive values were respectively 82.4% and 74.3% indicating that parallel assay of five TAAs raised the diagnostic precision greatly. Agreement rate and Kappa value were 76.9% and 0.52 respectively, which indicated that the observed value of this assay had middle range coincidence with actual value. The data from this study further support our previous hypothesis that detection of autoantibodies for diagnosis of certain type of cancer can be enhanced by using a mini-array of several TAAs as target antigens. In 19 colon cancer sera with carcinoembryonic antigen (CEA) negative, 11 (57.9%) were found to have anti-TAA antibodies. When CEA and anti-TAAs were used together as markers in colon cancer detection, the diagnostic sensitivity could be raised from 60.9% to 82.6%. A customized antigen mini-array using a panel of appropriately selected TAAs can enhance autoantibody detection in immunodiagnosis of colon cancer. Anti-TAA and CEA were independent markers and the simultaneous use of these two markers significantly raised the sensitivity of colon cancer detection.