Sera of cancer patients may contain antibodies that react with a unique group of autologous cellular antigenscalled tumor-associated antigens (TAAs). The present study aimed to determine whether a mini-array ofmultiple TAAs would enhance antibody detection and be a useful approach in esophageal cancer detection anddiagnosis. Our mini-array of multiple TAAs consisted of eleven antigens, p53, pl6, Impl, CyclinB1, C-myc, RalA,p62, Survivin, Koc, CyclinD1 and CyclinE full-length recombinant proteins. Enzyme-linked immunosorbentassays (ELISA) were used to detect autoantibodies against eleven selected TAAs in 174 sera from patients withesophageal cancer, as well as 242 sera from normal individuals. In addition, positive results of ELISA wereconfirmed by Western blotting. In a parallel screening trial, with the successive addition of antigen to a finaltotal of eleven TAAs, there was a stepwise increase in positive antibody reactions. The eleven TAAs were the bestparallel combination, and the sensitivity and specificity in diagnosing esophageal cancer was 75.3% and 81.0%,respectively. The positive and negative predictive values were 74.0% and 82.0%, respectively, indicating that theparallel assay of eleven TAAs raised the diagnostic precision significantly. In addition, the levels of antibodies toseven antigens, comprising p53, Impl, C-myc, RalA, p62, Survivin, and CyclinD1, were significantly different invarious stages of esophageal cancer, which showed that autoantibodies may be involved in the pathogenesis andprogression of esophageal cancer. All in all, this study further supports our previous hypothesis that a combinationof antibodies might acquire higher sensitivity for the diagnosis of certain types of cancer. A customized miniarrayof multiple carefully-selected TAAs is able to enhance autoantibody detection in the immunodiagnosis ofesophageal cancer and autoantibodies to TAAs might be reference indicators of clinical stage.