The role of amyloid-β (Aβ) neuropathology and its significant changes in biofluids after traumatic brain injury (TBI) is still debated. We used ultrasensitive digital ELISA approach to assess amyloid-β1-42 (Aβ42) concentrations and time-course in cerebrospinal fluid (CSF) and in plasma of patients with severe TBI and investigated their relationship to injury characteristics, neurological status and clinical outcome. We found decreased CSF Aβ42 levels in TBI patients acutely after injury with lower levels in patients who died 6 months post-injury than in survivors. Conversely, plasma Aβ42 levels were significantly increased in TBI with lower levels in patients who survived. A trend analysis showed that both CSF and plasma Aβ42 levels strongly correlated with mortality. A positive correlation between changes in CSF Aβ42 concentrations and neurological status as assessed by Glasgow Coma Scale (GCS) was identified. Our results suggest that determination of Aβ42 may be valuable to obtain prognostic information in patients with severe TBI as well as in monitoring the response of the brain to injury.