Abstract: Objective:To analyze the serological test results of hemolytic disease of newborn and explore the relationship between serological results and related factors such as the newborn blood type,gender and age as well as research the laboratory basis for diagnosis and treatment for hemolytic disease of newborn. Method:3 256 neonatal with high blood bilirubin were selected,blood type,irregular antibody screening,serological test results of hemolytic disease of newborn(hemolytic three tests)were conducted. Result:928 cases were diagnosed as hemolytic disease of newborn(HDN)in 3 256 cases of hyperbilirubinemia,the positive rate was 28.5%,among them ABO-HDN were 889 cases,accounting for 95.8%,Rh-HDN were 39 cases,accounting for 4.2%;the ABO-HDN positive rate were 52.8%,42.1%,0.9% of newborn with A,B and AB blood type,the difference of the incidence of ABO-HDN and the comparison of newborn blood type was statistically significant;the total bilirubin levels of A,and B blood type were (313.9±88.1)μmol/L and (342.2±108.8)μmol/L,the difference of severity of ABO-HDN and newborn blood type was statistically significant,the ABO-HDN positive rate were of male,female newborn were 54.1%,45.9%,the difference was statistically significant;the ABO-HDN positive rate of newborn with age ≤ 5 days newborn was higher than that of newborn with age>5 days;the ABO-HDN positive rate of direct antiglobulin test(direct antiglobulin),free antibody test(free)and antibody release test(bleeding)were 34.9%,79.0%,100.0%,the difference was statistically significant.Conclusion:Neonatal hemolysis mostly caused by ABO blood incompatibility,and mostly appear in the male and newborn of blood type A.But the severity of children with type B is higher.The accuracy of antibody release test would be higher than that of direct antiglobulin test and free antibody test for the diagnosis of HDN and The earlier inspection is the higher the positive detection rate is.