Objective: This study aimed to explore the clinical value of carboxyhemoglobin (COHb) measured by multi-wavelength CO-oximetry for the early auxiliary diagnosis of hemolytic disease of the fetus and newborn (HDFN) and prediction of the selection of clinical intervention strategies. Methods: A total of 310 neonates with hyperbilirubinemia were enrolled in this retrospective study. According to blood group incompatibility and clinical diagnosis, the subjects were divided into two groups: the hemolysis group, consisting of neonates with blood group incompatibility and clinically diagnosed HDFN, which was further subdivided into two subgroups: confirmed subgroup (113 cases, positive direct Coombs test (DAT) and/or antibody release test) and suspected subgroup (53 cases, only positive serum free antibody test); the control group included 144 neonates with negative results of three hemolysis tests and clinically excluded HDFN. Clinical data including birth weight, gestational age and age at diagnosis were collected. Laboratory indicators including COHb, hemoglobin (Hb), percentage of reticulocytes (Ret%), total serum bilirubin (TBIL) and results of three hemolysis tests were detected. We analyzed the predictive value of COHb for HDFN and its efficacy in evaluating the severity of hemolysis. Results: There were statistically significant differences in age at admission, gestational age and feeding patterns between the hemolysis group and the control group (P<0.05), while no significant differences were observed in birth weight and gender. Significant differences were found in admission TBIL, Hb, COHb and Ret% between the two groups (P<0.05). The median COHb level in the hemolysis group was higher than that in the control group (1.5% vs 1.2%, P<0.01). ABO-HDFN accounted for 96.39% of the hemolysis group. The COHb level of the confirmed subgroup was higher than that of the suspected subgroup (P<0.05). Higher COHb levels were correlated with more frequent phototherapy, higher rates of exchange transfusion and intravenous immunoglobulin (IVIG) use. The COHb cut-off value of 1.45% yielded a sensitivity of 70.48% and a specificity of 86.84% for diagnosing HDFN. Combined detection of COHb, Ret% and Hb improved the diagnostic sensitivity (74.43%) and specificity (87.61%). Conclusion: The COHb level was significantly higher in the hemolysis group than in the control group, and higher in the confirmed subgroup than in the suspected subgroup. Higher COHb levels were associated with greater requirements for clinical interventions. COHb can serve as a reference indicator for auxiliary diagnosis and severity assessment of HDFN.