Objective To establish and validate a standard method using flow cytometry (FCM)in human ABO blood group antibody (Ab) detection. Methods Sera samples from 52 blood donors were incubated with standardize red blood cells (RBCs), and anti-A/B antibody (IgM/IgG) was measured by indirect flow cytometry after adding secondary isotype-specific fluoresce labeled Ab. The results were compared with those by using ELISA reader. Results The changes of anti-A/B IgM measured by both methods correlated well and an overall correlation coefficiency of 0.730 for IgM was obtained by Spearman's rank testing (P＜0.05). FCM showed better specificity, sensitivity and reproducibility over ELISA reader. In blood group A samples (n = 16) or B samples (n = 16) ,the antirespectively. IgG blood group Ab could only be detected in blood group O sera. Conclusion Compared with traditional methods,FCM is a more objective method to offer accurate detection of natural ABO blood group Ab (IgM/IgG) and allowed semi-quantitative measurement of antibody levels.