Objective To investigate the clinical significance of human leukocyte antigen-B27 in ankylosing spondylitis(AS). Methods HLA-B27 of 854 patients suffering from AS, low back pain and/or arthritis were examined by immunomagnetic separation and fluoimmunoassay. Results The HLA-B27 positivity rate of 22 patients confirmed as AS was high (20/22, 90.9%) and they most were middle-young men. Conclusion HLA-B27 is closely connected to the occurrence of ankylosing spondylitis and can be used as an important auxiliary index for evaluating the diagnosis of AS.