〔Abstract〕 A high-throughput flow cytometry method was established to determine autoantibodies in the plasma of immune thrombocytopenia(ITP) patients and to explore the meaning of clinical significance. The monoclonal antibodies anti-platelet glycoprotein(GP) Ⅸ, Ⅰb, Ⅲa, Ⅱb and P-selectin were coated on five different strength APC-cy7 fluorescent microsbeads for flow cytometry detection. The receiver operating characteristic curve was established to evaluate the specificity and sensitivity of the method as well as the intra-assay CVs and inter-assay CVs differences. The intra-assay CVs were 8.35%, 5.65%, 3.41%, 3.63% and 3.51%, respectively. The inter-assay CVs were 10.10%, 9.13%, 8.04%, 9.59% and 10.20%, respectively. Sensitivity, specificity, and accuracy were 71.0%, 80.4% and 73.5% for anti-GPⅨ; 82.1%, 80.8% and 81.8% for anti-GPⅠb; 71.7%, 79.2% and 73.7% for anti-GPⅢa; 71.0%,76.7% and 72.5% for anti-GPⅡb; 72.0%, 79.2% and 74.0% for anti-P-selectin; 89.0%, 66.5% and 82.9% for the combined detection of the five antibodies. This method was used to detect the level of autoantibody before and after treatment in 75 ITP patients with oral dexamethasone. A new diagnostic method for platelet membrane glycoprotein autoantibodies in patients with immune thrombocytopenic purpura is successfully established based on flow microsphere technology.