A fast, high-throughput, automated multiple antigenic peptides microarray platform was established based on multiple peptide design and synthesis technology. Human cytomegalovirus(HCMV) glycoprotein B, phosphoprotein pp150 and Helicobacter pylori (Hp) urease (Ure) beta subunit were chosen as target protein. The advantageous linear epitope of these proteins was analyzed and screened. The multiple antigenic peptides(MAPs) containing the selected sequence were synthesized by Fmoc solid phase method,purified through high performance liquid chromatography(HPLC) and printed on nitrocellulose membrane in microarrays by computer-controlled high-speed robotics. The nitrocellulose membrane was blocked with 2% bovine serum albumin solution. The MAPs microarray was finished by assembling the membrane with plastic outer shell. Some microarrays were selected at random for quality control identified by control sera and compared with ELISA method. 4 MAPs were screened out, synthesized and identified. The result of positive and negative sera of Hp and HCMV detected by MAPs microarray were consistent with the control sera. In the clinical trial of 120 random sera, the microarray performed almost equally with ELISA method using recombinant antigen and microbial lysate antigen. The sensitivity and specificity of Ure-1, Ure-2 and PP150 MAPs were higher than 90%. The CV were lower than 7% among microarrays showing a good repeatability. It can be concluded that a MAPs microarray analytical platform which has a vast application prospect in assistant design of peptide vaccine was primarily established.