Background <p>Serum biomarkers are increasingly recognized as non-invasive ways of screening and prognostic evaluation for various medical conditions. The utility of ubiquitin C-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), and S100β in traumatic brain injury (TBI) needs detailed examination. The present work examined GFAP, UCH-L1, and S100β levels in TBI from mild to severe injuries.</p> Methods <p>Patients with a Glasgow Coma Scale (GCS) score of 3–15 were recruited, and plasma levels of UCH-L1, GFAP, and S100β were measured by ELISA.</p> Results <p>280 individuals participated in the study, comprising 140 healthy controls and 140 TBI patients. The mean ± standard deviation (SD) ages were 34.82 ± 3.14 years (controls) and 35.50 ± 11.05 years (TBI). In TBI patients S100β increased significantly from Day 0 at the time of admission with values of 1.86 ± 0.47 ng/mL, 1017.43 ± 56.31 pg/mL and 1.713 ± 0.31 ng/mL and decreased significantly by Day 7 of hospitalization with values of 0.266 ± 0.12 ng/mL, 267.98 ± 36.01 pg/mL and 0.05 ± 0.023 ng/mL, respectively (<i>p</i> &lt; 0.001). A significant decline in biomarker levels was observed over the one week in TBI patients.</p> Conclusions <p>This study demonstrates that serum levels of GFAP, UCH-L1, and S100β are strongly elevated in TBI patients and correlate significantly with the severity of injury as measured by the GCS. Furthermore, high initial levels of these biomarkers, particularly GFAP and UCH-L1, are predictive of unfavourable functional outcomes at discharge. These biomarkers provide valuable complementary prognostic information alongside traditional clinical assessments. Also, these biomarkers demonstrated strong evidence as a reliable marker of neuronal injury and associated morbidity risk that provided complementary prognostic value along with the GCS score.</p>