The present study aimed to explore the diagnostic value of the combination of cranial magnetic resonance imaging (MRI), serum homocysteine (HCY) and procalcitonin (PCT) for hyperbilirubinemia complicated with brain injury in neonates. One hundred and forty-nine children with hyperbilirubinemia admitted to Shandong Medical Imaging Research Institute from January 2014 to April 2016 were collected as research subjects, and were divided into a brain injury group (n=67) and a non-brain injury group (n=82) according to whether children suffered from brain injury. PCT levels were detected by electrochemiluminescence (ECL), and HCY levels by enzymatic cycling assay (ECA). The combination of cranial MRI, HCY and PCT was used to diagnose hyperbilirubinemia complicated with brain injury in neonates. The concentrations of HCY and PCT in the brain injury group were significantly higher than those in the non-brain injury group (P<0.001). According to the MRI examination results, the patients were divided into an MRI normal group and an MRI abnormal group. In the brain injury group, the serum HCY and PCT levels of the MRI abnormal group were significantly higher than those of the MRI normal group, with a statistically significant difference (P<0.05). In the non-brain injury group, the serum HCY and PCT levels of the MRI abnormal group were significantly higher than those of the MRI normal group, with a statistically significant difference (P<0.05). The sensitivity of the combined detection was significantly higher than that of single detection (P<0.05); the specificity was significantly higher than that of HCY detection (P<0.05), and the accuracy was significantly higher than that of MRI and HCY single detection (P<0.05). In conclusion, the combination of cranial MRI, HCY and PCT, which has a high diagnostic value for hyperbilirubinemia complicated with brain injury in neonates, is conducive to the early diagnosis and timely treatment of the disease and the reduction of sequelae.