Objective: To investigate the expression and clinical diagnostic value of Toll like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) in conjunctival epithelial cells and tears of patients with dry eye syndrome. Methods: From January 2023 to June 2024, 104 dry eye patients (disease group) who visited our hospital and 100 healthy individuals (control group) who underwent physical examination were selected. The changes of TLR4 and NF-κB in conjunctival epithelial cells and tears were analyzed. Pearson analysis was applied to analyze the correlation between TLR4 and NF-κB expression in conjunctival epithelial cells and tears. Logistic analysis was applied to analyze the factors that affected dry eye syndrome. ROC was applied to analyze the diagnostic value of TLR4 and NF-κB expression in conjunctival epithelial cells and tears for dry eye syndrome. Results: The expression levels of TLR4 and NF-κB in conjunctival epithelial cells and tears in the disease group were obviously higher than those in the control group (P<0.05). There was a positive correlation between TLR4 and NF-κB in conjunctival epithelial cells and tears (r=0.392, 0.348, P<0.05). The differences in the use of eye drops, BUT value, SIT value, TFT, and CFS scores between the disease group and the control group were statistically significant (χ2/t=9.525, 29.069, 38.498, 21.722, 27.487, P<0.05). Frequent use of eye drops, CFS score, TLR4, and NF-κB were risk factors for dry eye syndrome (OR=2.153, 3.183, 1.578, 2.452, P<0.05), while BUT value, SIT value, and TFT value were protective factors for dry eye syndrome (OR=0.654, 0.755, 0.276, P<0.05). The sensitivity, specificity, and AUC of TLR4 combined with NF-κB in conjunctival epithelial cells in the diagnosis of dry eye were 86.54%, 81.00%, and 0.889%, respectively. The combination of TLR4 and NF-κB in conjunctival epithelial cells had higher diagnostic value for dry eye than alone diagnosis (Z combination - TLR4=3.506, P=0.001, Z combination - NF-κB=3.165, P=0.002). The sensitivity, specificity, and AUC of TLR4 combined with NF-κB in tears for diagnosing dry eye syndrome were 82.69%, 70.00%, and 0.818, respectively. The combination of TLR4 and NF-κB in tears had higher diagnostic value for dry eye syndrome than alone diagnosis (Z combination - TLR4=3.117, P=0.002, Z combination - NF-κB=2.363, P=0.018). Conclusion: The expression levels of TLR4 and NF-κB in conjunctival epithelial cells and tears of patients with dry eye syndrome are elevated. TLR4 and NF-κB are related to the development of dry eye syndrome, and The increase in both can increase the risk of developing dry eye syndrome. The combination of TLR4 and NF-κB has certain diagnostic significance for dry eye syndrome.