Traumatic brain injury (TBI) can cause brain function loss injury-related deaths. Early TBI management is significant, but requires a timely, accurate, and sensitive assessment solution. However, current radiation- or imaging-based TBI diagnostic technologies are either less effective or time-consuming and expensive. Here, a surface-enhanced Raman scattering paper-based lateral flow strip (SERS-PLFS) is developed for rapid detection of neuron-specific enolase (NSE), a TBI biomarker in clinical blood plasma samples. The SERS-PLFS device is validated analytically with clinical human blood plasma samples. The performance metrics including accuracy, precision, reportable range, lower limit of quantification and upper limit of quantification, recovery, uncertainty, selectivity, robustness, are assessed using statistical methods. The validation results show that the SERS-PLFS is reliable in measuring the NSE levels in TBI patients.