A parallelled purification procedure suitable for acquiring high purified multiple recombinant antigen proteins should be established for construction of antigen protein microarray. An efficient approach for purification of tumor antigen proteins by affinity chromatograph and prepared gel electrophoresis for construction of tumor antigen microarray was established and evaluated. In the procedure, the inclusion bodies were prepared firstly. Then, a Ni-NTA His-bind resins was applied to all dissolved inclusions. The yield and purity of the affinity purified step were 71% and 70% respectively. After the initiative affinity chromatograph purification, the eluted purified recombinant antigen proteins were runned on the prepared scaled SDS-PAGE, and the purified recombinant antigen protein bands were indicated by KCl solution and cutted out. Furthermore, a common dialysis and refolding procedure were applied to the electro-eluted purified recombinant antigen protein solutions. The yield and purity of this further purified step were 32% and 95% respectively. Furthermore, the reaction of purified recombinant RPS4 antigen against the anti-RPS4 autoantibody in esophageal cancer patients' sera was confirmed by ELISA. The 16 purified antigen proteins were pin-printed on the aldehyde glass slides for construction a protein microarray. The reaction of the RPS4 antigen in the microarray against the anti-RPS4 autoantibody in esophageal cancer patients' sera was the same as that assayed in ELISA. The research suggests that affinity chromatograph combining prepared gel electrophoresis was an efficient parallelled purification approach for tumor antigens in construction of microarray.