Abstract: Similar to humans, most of non-human primates also contain the ABO blood group system. In this study, we sought to evaluate the ABO antibody (Ab) levels in monkeys using a modified flow cytometry method (FCM). The standard commercial human A or B-red blood cells (RBCs) were used as target cells. The binding of target cells and anti-A or B blood group Ab in sera of rhesus or cynomolgus monkeys was detected by flow cytometry after adding secondary specific fluorescence-labeled anti-human IgG or IgM Ab. Human healthy blood donor sera were used as controls. The results revealed that, using clear monkey sera, which were pre-absorbed on normal human type O RBCs to remove non-specific anti-human Abs, the modified FCM gave an accurate detection of ABO Ab levels in monkeys. When compared with the results of human sera, the distribution of ABO Ab levels in monkey sera were significantly lower (P<0.05). We concluded that the modified FCM could be used for the detection of monkey ABO Ab levels. The technique and data will be very valuable for the future establishment of ABO-incompatible organ transplant models in non-human primates, which could improve clinical applications.