Objective　To investigate the diagnostic value of urine S-100B protein levels in brain injury in premature infants. Methods A total of 76 premature infants with gestational age less than 35 weeks were selected from January to December 2012. Urine samples were obtained at 24 h, 72 h and 120 h after birth, and S-100B protein levels in urine were detected by chemiluminescence. According to the results of cranial ultrasound and MRI, all infants were divided into periventricular leukomalacia (PVL) group (n=16), periventricular-intraventricular haemorrhage (PVH-IVH) group (n=20) and no brain injury group (control group) (n=40). Urine S-100B protein levels were compared among groups. Results There was significant difference in Urine S-100B protein levels among PVL, PVH-IVH and control groups at 24 h, 72 h and 120 h after birth (P<0.01). At each time point, the urine S-100B protein levels were highest in PVL group and lowest in control group, and the differences were significant (P<0.01). The urine S-100B protein levels were significantly different among different time points in PVL and PVH-IVH groups (P<0.01), reaching a peak at 72 h and starting to decrease at 120 h. Conclusions Urine S-100B protein can be used as a sensitive indicator of brain injury in judging disease severity and assessing prognosis.