Background <p>Traumatic brain injury (TBI) is a leading cause of pediatric mortality and long-term morbidity. Hypoxia-inducible factor-1α (HIF-1α) and its downstream target, BCL2/adenovirus E1B 19&#xa0;kDa protein-interacting protein 3 (BNIP3), are key mediators of the hypoxia response and secondary brain injury. However, their temporal serum dynamics and clinical utility in pediatric TBI remain undefined. This study aimed to measure serum HIF-1α and BNIP3 levels at admission and day 7 post-TBI to evaluate their dynamic changes and relationship with injury severity and neurological outcome.</p> Methods <p>In this prospective single-center study, 169 pediatric TBI patients were enrolled. Serum HIF-1α and BNIP3 levels were quantified by ELISA at admission and day 7 post-injury. The relationship between biomarker dynamics (Δ, admission minus day 7), initial injury severity, and 6-month neurological outcome was analyzed.</p> Results <p>Admission levels of both HIF-1α and BNIP3 were associated with TBI severity. At day 7 post-TBI, serum HIF-1α decreased significantly in mild TBI, while BNIP3 showed a non-significant trend. In contrast, both biomarkers decreased significantly in the moderate-to-severe TBI group. Among children with moderate-to-severe TBI, a smaller reduction (Δ) in HIF-1α and BNIP3 levels over the first week was independently associated with unfavorable outcome after adjusting for the motor Glasgow Coma Scale score (ΔHIF-1α: aOR = 0.149, <i>P</i> = 0.029; ΔBNIP3: aOR = 0.297, <i>P</i> = 0.046). This association remained robust in a sensitivity analysis of survivors.</p> Conclusion <p>The early trajectories of serum HIF-1α and BNIP3 are independently associated with neurological outcomes in pediatric moderate-to-severe TBI. Serial measurement of these biomarkers may enhance risk stratification.</p> Trial registration <p>ChiCTR2100048753 (16/07/2021).</p>