Objective To establish a method for detection of platelet-associated autoantibodies against platelet-specific receptors by cytometric bead assay. Methods The detection beads were coated by monoclonal antibodies against platelet glycoprotein Ib, IIb, IIIa, and IIb/IIIa to capture platelet glycoprotein combined platelet-associated autoantibodies. Fluorescein isothiocyanate labeled polyclonal goat antihuman immunoglobulin antibody was added, and flow cytometer was used to detect bead-platelet-associated autoantibodies-antihuman immunoglobulin antibody complex. Results The fluorescene ratio of four monoclonal antibodies was significantly different (P <0.01) between the idiopathic thrombocytopenic purpura (ITP) patients and either the non-ITP patients or the normal controls. If the upper limit of normal control was set as cutoff value, ratios of greater than 1.37, 1.24, 1.48, and 1.19 were considered positive for the four monoclonal antibodies respectively. The flow cytometric bead assay had an overall sensitivity of 73.17% and a specificity of 94.29%. The overall sensitivity was significantly higher (P <0.05) than that of modified indirect MAIPA and that of using single antibody. Conclusions Platelet-associated autoantibodies can be detected rapidly by flow cytometric bead assay. Combined detection of autoantibodies can improve the positive rate. The assay would be valuable for diagnosis of ITP.