Objective To analyze the serological detection results of hemolytic disease of newborns(HDN)in neonatal hyperbil-irubinemia and to explore the relationship between serological results and related factors.Methods The neonatal hemolytic three i-tems test were performed on blood specimens of 796 cases of neonatal hyperbilirubinemia.The relationship between the positive rate of HDN with the related factors such as blood type,specimens collecting time,hemoglobin level and serum total bilirubin value was analyzed.Results Among 796 cases of hyperbilirubinemia,184 cases were HDN(23.12%),in which 796 cases were ABO-HDN and 8 cases were Rh-HDN.The direct free antibody test,antibody release test and direct antiglobulin test were positive in 180 cases (22.61%),184 cases(23.12%)and 140 cases(17.59%)respectively.The HDN positive rate of the newborns with B blood type was higher than that of the newborns with A blood type,the difference between them had statistical significance(χ2 = 10.304,P <0.05).The HDN positive rate was closely related with the specimen collection time,moreover,the greater the day age,the higher the HDN positive rate,the difference showing statistical significance(P <0.05).The hemoglobin level and serum total bilirubin val-ue had statistically significant difference between newborns with HDN and those without HDN(P <0.05).Conclusion The posi-tive rate of HDN is high in newborns with hyperbilirubinemia.The hemolytic three items test should be performed as early as possi-ble.Combining with the hemoglobin level and serum total bilirubin value can increase the HDN detection rate.