〔Abstract〕 Objective To investigate the diagnostic value of amplitude integrated electroencephalogram combined with serum hypoxia inducible factor-1α (HIF-1α) and neurofilament protein (NFP) in neonatal brain injury. Methods A total of 76 full-term neonatal hypoxic-ischemic encephalopathy (HIE) admitted to the First People's Hospital of Lingbao from January 2020 to January 2023 were selected as the observation group. According to the degree of brain injury, they were divided into mild injury group ( 19 cases ), moderate injury group ( 37 cases ) and severe injury group ( 20 cases ). The amplitude-integrated electroencephalography (aEEG) monitoring and serum HIF-1 α and NFP levels were detected. And 50 healthy newborns were selected as the control group. The receiver operating characteristic (ROC) curve was used to evaluate the value of aEEG, HIF-1 α, NFP and the combined diagnosis of neonatal brain injury. Results The levels of serum HIF-1α and NFP in the observation group were significantly higher than those in the control group (P < 0.05). With the aggravation of brain injury, the levels of serum HIF-1α and NFP increased (P < 0.05). ROC curve analysis showed that the area under the curve (AUC) of serum HIF-1α in the diagnosis of neonatal HIE was 0.683 (95 % CI : 0.327-0.973), and the cut-off value was 764.09 pg·mL^-1. The AUC of serum NFP in the diagnosis of neonatal HIE was 0.771 (95 % CI : 0.506-0.879), and the cut-off value was 19.29 pg·mL^-1. The AUC of aEEG, serum HIF-1α and NFP in the diagnosis of neonatal HIE was 0.889 (95 % CI : 0.627-0.995). Conclusion The combination of aEEG and serum HIF-1α and NFP has high potential in the diagnosis of neonatal HIE.