We selected 12 antigens corresponding to commonly used autoantibodies in clinical practice to prepare antigen microarray. We chose NBT/BCIP color reaction as the end detection strategy to develop a new autoantibody protein chip detection system. Using this system, we optimized the best spotting solution, spotting concentration of the 12 antigens and the dilution of serum. We prepared a protein chip that could detect 12 autoantibodies simultaneously using the optimized antigen concentration. We established a new method to determine the cutoff of each autoantibodies by evaluation of 678 positive and 120 negative serum of clinical sample. We also evaluated the sensitivity and specificity of our new detection system. The optimal spotting solution was 0.1% TBST, the dilution of serum was 1:4 and the best spotting concentration of the 12 antigens were ANA 520 μg/mL, Ro-60/SSa 465 μg/mL, La/SSb 530 μg/mL, Jo-1 530 μg/mL, Scl-70 525 μg/mL, Sm 520 μg/mL, Ro-52/SSa 615 μg/mL, RF 340 μg/mL, CCP 465 μg/mL, u1RNP 410 μg/mL, CENP-B 490 μg/mL and dsDNA 580 μg/mL respectively. It had higher coincidence rate compared to current clinical used methods. We have developed a 12 antigens protein chip for the detection of autoantibodies based on the NBT/BCIP color reaction system. This system was fast, convenient, efficient, and cost-effective.