Objective To explore the application value of diffusion-weighted imaging (DWI) combined with serum neuron specific enolase (NSE) in the diagnosis of punctate white matter lesions in premature infants. Methods A total of 92 premature infants with punctate white matter lesions (included in observation group) and 35 premature infants born without punctate white matter lesions (included in control group) in our hospital from May 2016 to May 2018 were selected for the study. The two groups were given DWI scanning to detect the serum NSE levels on the day of birth and at the 1st w and 2nd w of birth in the two groups. The children patients in observation group were divided into mild group, moderate group and severe group according to the severity of disease. The serum NSE levels on the day of birth and at the 1st w and 2nd w of birth were compared among the three subgroups. The ROC curve was used to evaluate the efficacy of DWI combined with serum NSE in the diagnosis of punctate white matter lesions in premature infants. Results The NSE level at the time points after birth was successively decreased significantly in the two groups, and the NSE level in observation group on the day of birth and at the 1st w and 2nd w of birth was significantly higher than that in control group (P<0.05). With the exacerbation of severity of punctate white matter lesions in premature infants, the serum NSE level was significantly increased (P<0.05), and the NSE was successively decreased significantly in mild group, moderate group and severe group on the day of birth and at the 1st w and 2nd w of birth (P<0.05). The sensitivity, specificity, accuracy and AUC of DWI combined with serum NSE in the diagnosis of punctate white matter lesions in premature infants were significantly higher than those of DWI and serum NSE (P<0.05). Conclusion DWI combined with serum NSE has clear application value in the diagnosis of punctate white matter lesions in premature infants. The combination of the two can be used as an effective means to diagnose punctate white matter lesions in premature infants and to assess the severity of brain injury.