【Objective】 To investigate the value of three hemolysis tests and carboxyhemoglobin (COHb) level in the diagnosis of hemolytic disease of the fetus and newborn (HDFN). 【Methods】 From January 1, 2019 to December 31, 2022, the neonates hospitalized in the Department of Neonatology of Hebei Provincial Children's Hospital with suspected hemolytic disease who had serological testing were retrospectively enrolled in the study. They were distributed into HDFN group and non-HDFN group according to the final diagnosis. Their clinical and laboratory data were collected and analyzed, and the COHb level was detected by blood gas analyzer. 【Results】 A total of 378 neonates with HDFN and 217 neonates without HDFN were included in the study. Most of the neonates in HDFN group were full-term infants (348/378, 92.1%), with median gestational age of 39.1 (38.3, 40.0) weeks. Three hundred and fifty-four cases (354/378, 93.7%) were ABO-HDFN and the rest were Rh HDFN. There were significant differences in the level of serum total bilirubin, hemoglobin, COHb and reticulocyte percentage at admission between the two groups(P<0.05). The positive rate of three hemolysis tests in HDFN group decreased with the increase of the days after birth. The highest positive rate (more than 80%) was observed within 2 days after birth. Correlation analysis showed a negative relationship between the COHb level and the age (rs = -0.434, P<0.001) . Among the three hemolysis tests in HDFN group, the positive rate of antibody release test was the highest (69.0%), followed by the free antibody test (55.6%) and the direct antiglobulin test (DAT) (36.0%). Receiver operating characteristic (ROC) curve analysis showed that the optimal cut-off value of COHb was 1.15%.The sensitivity of COHb ≥ 1.15% was 51.8%, higher than single DAT (36.0%) . The diagnosis effectiveness of three hemolysis tests couldn't be improved when combined with COHb detection(Z = -0.727, P>0.05) . 【Conclusion】 The three hemolysis tests are important in the diagnosis of HDFN, among which the antibody release test has the highest sensitivity. COHb has certain value for the diagnosis of HDFN, but joint testing cannot improve the diagnosis effectiveness of three hemolysis tests. Hemolysis tests and/or COHb detection should be conducted for neonates at risk of hemolysis as early as possible after birth.