Objective: To analyze the results of irregular antibody detection in early and late pregnancy and to study its clinical significance and safety strategy. Methods: A total of 14 770 pregnant women visited Shanghai First Maternity and Infant Hospital from July 2018 to September 2019 were enrolled and detected for irregular antibodies in the first trimester (≤16 weeks) and late pregnancy (≥32 weeks). In pregnant women with positive irregular antibody, umbilical cord blood or venous blood of newborn infant were collected for the testing of hemolytic disease of fetus and newborn HDFN. Results: Of the 14 770 case of pregnant women, 63 cases had irregular antibodies detected in the early pregnancy, with a positive rate of 0.43%, while 97 cases had irregular antibodies detected in late pregnancy, with a positive rate of 0.66%( χ²=7.264, P< 0.05). The number of cases with irregular antibody detected increased by 34cases in late pregnancy, accounting for 35.05% of the total positive irregular antibody cases. Majority of irregular antibody detected were anti-M antibodies, accounting for 33.33% and 34.02% in early and late pregnancy, respectively. Anti-E antibodies accounted for 12.69% and 16.49%, respectively, while proportion of anti-D antibodies were 7.94% and 8.25%, respectively. The distribution of antibody types between early and late pregnancy was similar( χ²=2.269, P>0.05). Of the 63 cases with irregular antibodies detected in early pregnancy, the titration of antibody increased by 2 gradients in 11 cases at late pregnancy, and 7 of them reached ≥ 64. The 34 cases with irregular antibody newly detected in late pregnancy generally had lower titration of antibodies, with only 1 case with titration≥64. Amongthe fetus delivered by 97 pregnant women with irregular antibodies, 12 were confirmed having non-ABO-HDFN; 8 were delivered by pregnant women with irregular antibody detected at early pregnancy, and 6 of the 8 were of the RH system (anti-D5, anti-Ec1), with more severe symptoms requiring blood transfusion or exchange transfusion. The 4 fetuses with non-ABO-HDFN delivered by women with irregular antibody newly detected at late pregnancy had mild symptoms. Conclusions: The detection rate of irregular antibody in late stage of pregnancy in Shanghai is obviously higher than that in early stage of pregnancy. Higher titer of antibody in early stage of pregnancy may result in serious HDFN. Retesting of irregular antibody in late pregnancy has some significance for ensu-ring the safety of mother and child.