Objective: To investigate the effects of blood transfusion and number of pregnancies on ABO antibody titers and irregular antibodies in pregnant women with type O blood. Materials and Methods: The study included 4,200 pregnant women with type O blood (their husbands were with non-O type blood) that were divided into transfusion group and non-transfusion group, according to whether they had a history of blood transfusion. The both groups were respectively divided into three subgroups (the number of pregnancies was one, two, and ≥ three). The ABO antibody titers and irregular antibodies were detected at the same time. The effects of ABO antibody titers and irregular antibodies on hemolytic disease of the newborn (HDN) were discussed. Results: There was no consistency of ABO antibody titers and existence of irregular antibody. The positive rates of irregular antibody of transfusion group and of the subgroup (number of pregnancies ≥ three) were far higher than that of non-transfusion group and of the subgroups (number of pregnancies < three), respectively. All pregnant women with positive irregular antibody in non-transfusion group were with HDN. Conclusions: For pregnant women with number of pregnancies ≥ three or with history of blood transfusion, the prenatal joint detection of ABO antibody titers and irregular antibodies is helpful for accurately reflecting the in vivo antibody type and level.