Objective: To explore the relationship between serum NGF and NF-L levels with the severity and prognosis of traumatic brain injury (TBI) patients, as well as their predictive value, a prospective research was performed. Methods: 132 TBI patients and 132 ordinary trauma patients were collected as the observation group and control group, respectively. TBI patients in the observation group were divided into three subgroups of mild, moderate, and severe according to GCS scores, and into good and poor prognosis subgroups according to GOS scores at 6-month follow-up. ELISA was used to detect serum NGF and NF-L levels in all patients after admission. Through comparison between the observation and control groups, the relationship between TBI and serum NGF and NF-L levels was clarified. By avoiding differences between subgroups, the relationship between serum NGF and NF-L levels and the severity and prognosis of TBI were determined. Through correlation analysis, ROC curve analysis and multiple factor logistic regression analysis, the predictive value of serum NGF and NF-L levels for TBI was clarified. Results: Compared with the control group, serum NGF and NF-L levels were significantly increased while GCS scores were significantly decreased in the observation group, P<0.01. In addition, serum NGF and NF-L levels gradually increased among subgroups of mild, moderate, and severe TBI patients in the observation group, with significant differences, P<0.01. The AUC of serum NGF and NF-L levels for predicting mild TBI were 0.924 and 0.991, and the optimal cut-off points were 12.5 ng/mL and 66.5 pg/mL, respectively. The AUC of serum NGF and NF-L levels for predicting moderate TBI were 0.776 and 0.837, respectively, and the AUC for predicting severe TBI was 0.950 and 0.988, respectively, with optimal cut-off points of 14.5 ng/mL and 157.0 pg/mL, respectively. Spearman correlation analysis showed that serum NGF and NF-L levels were negatively correlated with GOS scores of TBI patients, with P<0.01. The AUC for predicting the prognosis of TBI patients with serum NGF and NF-L levels were 0.858 and 0.909, respectively, with optimal cut-off points of 13.5 ng/mL and 71.5 pg/mL, respectively. Logistic regression analysis showed that serum NGF and NF-L levels were risk factors affecting the prognosis of TBI patients. Conclusion: Serum NGF and NF-L levels are significantly elevated in TBI patients and can provide reference for early assessment of injury severity in clinical practice. Furthermore, elevated serum NGF and NF-L levels are associated with poor prognosis of TBI patients and are good biological indicators for predicting prognosis.