Objective To explore the clinical significance of serum and urine S-100B at different time points after birth in evaluating the severity and predicting the prognosis of neonatal hypoxic-ischemic encephalopathy (HIE). Method Serum and urine S-100B were measured in the 48 HIE neonates and 25 control neonates at day 1,3 and 7 after birth by ELISA and followed up at age of 1 year for HIE infants. Results The levels of serum and urine S-100B at day 1,3 and 7 after birth in the HIE group were significantly higher than in control group (P<0.01 or <0.05) and there was a close positive relationship between the level of S-100B protein and severity of HIE.The levels of serum S-100B at day 1 and 3 and urine S-100B at day 3 and 7 after birth in the abnormal group of outcome were significantly higher than normal group of outcome at age of 1 year (P<0.05 or <0.01).At a cut off >5 μg/L of serum S-100B protein and >3 μg/L of urine S-100B in there was a relatively good predictive value of S-100B for neonatal HIE at age of 1 year.The accuracy of serum S-100B>5 μg/L at day 1 and 3 after birth for predicting the prognosis of neonatal HIE was 88.1% and 77.3%,respectively and the accuracy of urine S-100B>3 μg/L at day 3 and 7 after birth for predicting the outcome of neonatal HIE was 90.5% and 85.7%,respectively.There was no significant difference between serum and urine S-100B for predicting the prognosis of neonatal HIE. Conclusions There is a close relationship between the level of serum and urine S-100B and the severity and the prognosis of neonatal HIE.S-100B protein shows significant value in predicting the prognosis of neonatal HIE.