Abstract: Objective To evaluate the application of serum ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) and heme oxidase-1 (HO-1) combined with amplitude integrated electroencephalogram (aEEG) for early diagnosis and prognosis of brain injury in preterm infants (BIPI). Methods A total of 100 preterm infants who were admitted to the neonatal intensive care unit (NICU), Northern Jiangsu People's Hospital from October 2021 to October 2022 were selected. According to the results of brain magnetic resonance examination, the preterm infants were divided into three groups:a control group (without abnormal results of brain magnetic resonance examination, n=40), a periventricular intraventricular hemorrhage (PVH-IVH) group (n=35) and a brain white matter injury (WMD) group (n=25). According to the development quotient (DQ) of Gesell development diagnosis scale corrected for gestational age of six months, premature infants in the PVH-IVH and WMD groups were further divided into two groups:a good prognosis group (n=45) and a poor prognosis group (n=15). The levels of serum UCH-L1 and HO-1 in the three groups were measured 24 and 72 h after delivery. Their scores of aEEG were compared 7 days after delivery and at 40 weeks of corrected gestational age. A ROC curve was plotted to evaluate the value of UCH-L1, HO-1 and aEEG scores in the early diagnosis and prognosis of BIPI. Results The levels of serum UCH-L1 and HO-1 in the PVH-IVH group and the WMD group were higher than those in the control group at 24 h and 72 h after delivery, where the WMD group had higher serum UCH-L1 and HO-1 levels than the PVH-IVH group (P<0.05). The PVH-IVH group and the WMD group showed significantly lower aEEG scores than the control group 7 days after delivery and at 40 weeks of corrected gestational age (P<0.05), while there was no statistical difference in aEEG score between the WMD group and the PVH-IVH group (P>0.05). The AUCs of UCH-L1, HO-1 and aEEG for early diagnosis of BIPI were 0.877, 0.889 and 0.781 respectively, whereas the AUC of UCH-L1 and HO-1 combined with aEEG was 0.929. The levels of serum UCH-L1 and HO-1 levels in the poor prognosis group were higher than those in the good prognosis group 24 h and 72 h after delivery (P<0.05). The poor prognosis group showed remarkably reduced aEEG score, compared with the good prognosis group 7 days after delivery and at 40 weeks of corrected gestational age (P<0.05). The AUCs of UCH-L1, HO-1 and AEEG for evaluating the prognosis of BIPI patients were 0.830, 0.861 and 0.782, respectively, whereas the AUC of UCH-L1 and HO-1 combined with aEEG was 0.953. Conclusions Preterm infants with brain injury show abnormal levels of UCH-L1 and HO-1 and aEEG scores, with certain value for early diagnosis and evaluation of prognosis.