Objective To explore and analyze the value of serum testing in the diagnosis of hemolytic disease in newborns with hyperbilirubinemia.Methods Sixty newborns with hyperbilirubinemia admitted to Xinxiang Maternal and Child Health Hospital from December 2021 to December 2022 were selected and subjected to three neonatal hemolytic disease serological tests(direct antibody test,dissociation test,and release test).The blood type of the children was identified using a blood type test card,and the serum test results and incidence of different blood types were compared.Results The experimental results showed that out of 60 newborns with hyperbilirubinemia,a total of 28 children tested positive(46.7%),4 children were suspected(6.7%),and 28 children were negative(46.7%).The total detection rate of hemolytic disease is 46.7%.Among them,12 cases were positive in the direct antibody test,with a positive detection rate of 42.9%,21 cases were positive in the free test,with a positive detection rate of 75.0%,and 28 cases were positive in the release test,with a positive detection rate of 100%.The positive detection rate in the release test was significantly higher than that in the direct antibody test and free test,and the difference was statistically significant(x2=22.401,P<0.001;x2=8.002,P-0.005);Among the 28 children with positive test results,there were 10 cases of type A blood,accounting for 35.7%,12 cases of type B blood,accounting for 42.9%,3 cases of type O blood,accounting for 10.7%,and 3 cases of type AB blood,accounting for 10.7%.The chi square test results showed that the probability of hemolytic disease in children with type A and B hyperbilirubinemia was significantly higher than that in children with type AB and O blood,with significant differences(x2=4.909,P=0.027;x2=3.776,P=0.007).Conclusion After the occurrence of hyperbilirubinemia in newborns,serum testing can effectively detect hemolytic disease,which has high clinical value and significance.