To study the expressions of glial fibrillary acidic protein (GFAP), Neuron-specific en ase (NSE) combined with S100β in patients with sepsis-associated encephalopathy (SAE) and their diagnostic value. Methods: A total of 186 sepsis patients admitted to the ICU of our hospital from January 2019 to January 2022 were selected and divided into the SAE group of 86 cases and the non-SAE group of 100 cases according towhether the patients had SAE or not. The clinical data (general data, laboratory indicators) of the two groups were collected, serum GFAP, NSE, And S100β levels were determined, and the degree of brain injury was evaluated according to the Glasgow Coma Scale (GCS), and the 28-day prognosis and survival of the patients were analyzed. The ROC curve was used to analyze the predictive value of GFAP, NSE and S100β on the occurrence of the same. Logistic regression was used to analyze the factors affecting the occurrence of SAE. Results: The levels of GFAP, NSE and S100β were higher than those in the non-SAE group (P<0.05). Logistic regression analysis showed that combined hyperuricemia, high Hcy, high SOFA score, high APACHE II score, high PCT level, high CRP level, high GFAP level, high NSE level, High S100β level was associated with the occurrence of SAE (P <0.05). The 28-day mortality in the SAE group was higher than that in the non-SAE group [31.40% (27/86) vs 10.00% (10/100)] (χ2=13.282, P < 0.05). With the aggravation of brain injury in SAE patients, the levels of GFAP, NSE, And S100β gradually increased. Pearson correlation analysis showed that the levels of GFAP, NSE and S100β were correlated with the degree of brain injury in SAE patients (P<0.05). The levels of GFAP, levels of GFAP, NSE and S100β in SAE death patients were higher than those in surviving patients (P<0.05) showing that the levels of GFAP, NSE and S100β were associated with the 28-day prognosis of SAE patients (P<0.05). The ROC curve showed that GFAP, NSE combined with S100βhad better diagnostic value for SAE (AUC=0.934, 95%CI=0.796-0.962, P < 0.05) Pearson correlation analysis showed that GFAP was positively correlated with NSE (r=0.453, P<0.05). GFAP was positively correlated with S100β (r=0.443, P < 0.05); NSE was positively correlated with S100β (r=0.620, P < 0.05). The occurrence of SAE is affected by a variety of factors. GFAP, NSE, and S100β are elevated in this disease and are related to the degree of brain injury and prognosis of patients. When the level is ≥0.56 mg/L, it indicates a higher risk of SAE and can be used for early diagnosis of this disease.