Objective To optimize several key factors in preparing protein microarray,and establish microarrays to detect autoantibodies.Methods Four factors,including incubation time of the microarray after spotting,the blocking reagent,the length of blocking time,the length of time conjugating with the second-an- tibody,were selected to carry out orthogonal optimization,then we determined the optimal spotting concentra- tion of antigen and the best dilution of serum with the optimized conditions obtained from the above.On the basis of the optimized conditions,we prepared microarray to detect six autoantibodies simultaneously,which were compared with IIF and ELISA.Results The best conditions we determined from the experiment were: incubating the spot microarray for 120 min,PBST containing 1% BSA and 2.5% saccharose as the blocking reagent and blocking for 30 min,reacting with the second-antibody for 30 min,the appropriate spotting con- centrations of six antigens differed from 100?g/ml to 300?g/ml and the best serum dilution was 1:2.Com- pared with IIF for ANA detection,the sensitivity of the microarray was 88.6%,the specificity was 83.3%,and compared with ELISA in detecting the other five autoantibodies,the sensitivity of the microarray was between 80.0% and 88.2%,and the specificity was between 90.2% and 98.6%.Conclusion The optimization condi- tions we obtained are suitable for preparing protein microarray,and the detection sensitivity and specificity of autoantibodies by microarray are consistent with the conventional methods in general.The microarray for de- tecting autoantibodies is applicable in the clinical diagnosis of autoimmune disease.