Objective: To investigate the correlation between the color Doppler ultrasonography of the brain of premature infants and the score of neonatal behavioral neurological assessment (NBNA) and brain injury. Methods: A total of 280 premature infants born in our hospital from June 2018 to January 2020 were selected as the study subjects. All premature infants underwent brain ultrasound examination on the 5th day of birth, and NBNA scores were performed on the 3rd, 7th, 14th and 28th day after birth. The occurrence and severity of brain injury in premature infants during hospitalization were analyzed according to the Diagnostic Criteria for Ischemic Hypoxic Encephalopathy of Newborns, the premature infants with brain injury were divided into a <31 weeks group, a 31–35 weeks group and a >35 weeks group according to gestational age, and the brain color Doppler ultrasound performance, NBNA score and their correlation with brain injury were analyzed. Results: The results of craniocerebral ultrasonography showed that a total of 79 cases of 280 premature infants had brain injury, and the incidence of brain injury was 28.21%, including 46 cases of grade I–II, 17 cases of grade III and 16 cases of grade IV. The NBNA scores of premature infants with brain injury at different gestational ages were significantly different at different time points (P<0.05); the NBNA scores of premature infants with brain injury of different gestational ages increased with the increase of age, and the differences were statistically significant (P<0.05). The results of craniocerebral ultrasonography showed a high consistency with the occurrence and severity of brain injury during hospitalization (Kappa value =0.802, P<0.001). The NBNA scores of grade I–II, III and IV premature infants were significantly lower than that in the group without brain injury, the NBNA score of premature infants in grade III and IV groups were significantly lower than that in grade I–II groups, and NBNA score of premature infants in grade IV group was significantly lower than that in grade III group, the difference were statistically significant (P<0.05). The results of linear regression analysis showed that NBNA score was negatively correlated with brain injury grade (P<0.05); the receiver operating characteristics (ROC) curve analysis showed that the area under the curve (AUC) of NBNA score was 0.839, the sensitivity was 87.14%, and the specificity was 79.64%. Conclusion: The brain color Doppler ultrasonography and NBNA score of premature infants are correlated with brain injury to some extent, the brain color Doppler ultrasonography can effectively assess the severity of brain injury in premature infants, and NBNA score can early diagnose the occurrence of brain injury in premature infants.