Autoantibody (autoAb) response is an important arm of endogenously arising anti-tumor immune responses， and has received new attention as a cancer biomarker with the recent success of immune check-point inhibitor therapy. Our laboratory has been focusing on measuring autoAb against B-cell epitopes in order to bypass the necessity to purify a panel of recombinant proteins. In order to optimize peptide-based autoAb measurement and to increase sensitivities to cover more patients， we developed a new approach of using mixed peptides to conjugate on the same microsphere and compared its results with the use of a dominant peptide epitope using Luminex microbead-based multiplex assays. The peptide epitopes of two cancer/germline antigens， New York esophageal cancer antigen-1 (NY-ESO-1) and X antigen family member-1b (XAGE-1b)， and cancer/stem cell antigen， sex determining region Y-box-2 (SOX2)， were used as prototypes in this study. Our results indicate that using mixed peptides of B-cell epitopes improves the sensitivity of detecting more patients with autoAb responses. Thus， when the full-length protein is not available for conjugating onto microspheres， a mixture of B-cell epitopes is the method of choice for using Luminex multiplex assay to detect autoAb response in cancer patients.