Objective Toinvestigate the value of the serum levels of high-mobility group box B1 (HMGB1) and glial fibrillary acidic protein (GFAP) in predicting the prognosis of neonatal hypoxic-ischemic encephalopathy (HIE).Methods A total of 115 children with HIE who were admitted to Danzhou People's Hospital were enrolled, and according to their prognosis, they were divided into survival group with 87 children and death group with 28 children. According to the clinical classification of neurological symptoms, they were divided into mild-moderate group with 80 children and severe group with 35 children. Serum levels of HMGB1 and GFAP on days 1, 3, and 5 were compared between groups. The receiver operating characteristic (ROC) curve was used to analyze the value of serum HMGB1 and GFAP levels in predicting death in children with HIE.Results Compared with the survival group on days 1, 3, and 5, the death group had significantly higher serum levels of HMGB1 (9.35±3.24/13.60±4.18/18.64±6.95 μg/L vs 5.80±2.16/6.12±2.28/4.70±1.82 μg/L, P<0.05) and GFAP (152.80±44.72/186.24±53.40/227.50±64.38 ng/L vs 103.60±31.52/120.38±34.75/115.60±32.42 ng/L, P<0.05), and the serum levels of HMGB1 and GFAP tended to increase over time in the death group (P<0.05). Compared with the mild-moderate group on days 1, 3, and 5, the severe group had significantly higher serum levels of HMGB1 (9.14±3.15/12.75±3.84/16.90±6.42 μg/L vs 6.38±2.37/6.63±2.40/5.82±2.14 μg/L, P<0.05) and GFAP (145.20±40.83/172.80±48.63/213.80±62.42 ng/L vs 112.73±33.60/131.46±36.72/24.73±34.58 ng/L, P<0.05), and the serum levels of HMGB1 and GFAP tended to increase over time in the severe group (P<0.05). The ROC curve analysis showed that the serum levels of HMGB1 and GFAP on day 3 had the largest area under the ROC curve of 0.926 (95% confidence interval[CI]:0.862-0.974), with a sensitivity of 93.0% and a specificity of 87.3%. The correlation analysis showed that serum HMGB1 level was positively correlated with serum GFAP level in the death group (r=0.806, P<0.01).Conclusions Increases in the serum levels of HMGB1 and GFAP are associated with disease severity in children with HIE, and the serum levels of HMGB1 and GFAP on day 3 have a high value in predicting the prognosis of children with HIE.