Circulating autoantibodies have a close association with autoimmune diseases, which may be seen even in healthy individuals. These are also considered as promising source of new biomarkers in various autoimmune diseases. However, their profile is not completely understood till now. Here, we evaluated autoantibodies against nuclear mitotic apparatus protein located at the carboxy terminus (C-NuMA)in blood circulation of Han Chinese patients, using different technical approaches to discover pathological reaction leading to Behçet's disease (BD). In the first step, the recombinant human carboxy-terminal region of NuMA peptide (C-NuMA) was over-expressed and purified. In the second step, the indirect immunofluorescence method was used with patients' sera, and commercial anti-NuMA antibody was used to determine the NuMA as a potential autoantigen. Results were confirmed at cell level by western blots, indicating that two of ten patients with Behçet's disease could react with the recombinant C-NuMA,and the presence of antibodies were further verified by immunoprecipitation technique. Finally, the corresponding immunoassay (ELISA) was developed and optimized with specific recombinant C-NuMA as an in vitro method to test the confirmed patients with Behçet's disease. Our findings demonstrated that C-terminus of NuMA is an immune target of Behçet's disease in Han Chinese patients.