Abstract: Objective:To examine the neonatal hemolytic three tests in 1 003 cases of neonatal hemolytic disease in Changzhou in order to study the distribution of specimens, and the relationship between the factors in blood type, sex, days of birth and so on.Method:We detected ABO blood group identification, Rh blood group identification and the neonatal hemolytic three tests on 1 003 neonatal hemolytic specimens from Changzhou hospitals in 2008-2016. The neonatal hemolytic three tests conducted red blood cells direct antiglobulin test (direct test), free antibody test (free test) and antibody release test (release test). The results of the tests were the direct indicators of neonatal HDN, and the statistical analysis was performed according to blood type, sex, days of birth and so on.Result:The main causes of neonatal hemolytic disease were the antibodies of ABO system and Rh system which brought on hemolysis. The strength of the result on the direct test was an important sign to distinguish between ABO and Rh system hemolysis. The result was more than 2 plus indicating the hemolysis mainly caused by Rh system antibody, while less than 2 plus often indicating the hemolysis of ABO system. The occurrence probability of ABO hemolysis was much higher than that of Rh hemolysis, mainly in type A and type B blood, and the incidence of HDN in type A blood was significantly higher than that of type B. The incidence of Rh-HDN that mostly caused by antibody-D was significantly lower than that of ABO-HDN. The incidence of HDN in female children was significantly higher than that in male children. The positive rate of HDN was significantly higher within 7 days than that more than 7 days after birth. Conclusion:The diagnosis of neonatal HDN was confirmed by the neonatal hemolytic three tests, in which the results of the direct test could be provided to laboratory staff to distinguish ABO and Rh hemolytic disease. Especially in the emergency process, under the premise of a clear medical history and in combination with patients with bilirubin indicators, the staff could make a rapid analysis and determine whether the need for newborns blood transfusion. It would be of great significance for the clinical rescue. In the detection process, we should also pay attention to the patient's gender, age, blood and other factors. Once suspected as HDN, the newborns should be collected of blood samples for inspection as soon as possible (within 7 days of birth).