Objective To establish and optimize flow cytometry method for the detection of human leukocyte antigen B27 (HLA B27) and compare it with serological method. Methods A total of 98 samples from blood donors were entered into the study. HLA B27 was detected with ethylenediaminetetra aceticacid(EDTA), heparin sodium and different dosages of B27 monoclonal antibody, respectively. Other 30 samples were typed by microlymphocytotoxicity test(serological method). Results Genotyping for HLA B27 was successfully performed in 98 samples by flow cytometry, containing 2 cases with B27 positive, 96 cases with B27 negative. The types of anticoagulant agents and dosages of monoclonal antibody(7～30 ?l) had no remarkable effect on the results. The results of serological method for the detection of 30 samples were consistent with those by flow cytometry method. Conclusion Genotyping for HLA B27 by flow cytometry is a rapid and accurate technique with high sensitivity, suitable for clinical application. Detection of HLA B27, a very important basis for the diagnosis and differential diagnosis of ankylosing spondylitis(AS) and other joint diseases of the vertebral column, is of a remarkable clinical significance.