Objective To evaluate the expression level of nuclear factor kappa B (NF-κB) in patients with Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL), and to examine its impact on patients’ clinical characteristics and prognosis. Methods Patients with EBV-positive DLBCL who were diagnosed and treated at West China Hospital of Sichuan University between January 2010 and December 2020 were selected as the research subjects. Clinical data were retrospectively collected. According to the expression of NF-κB, the patients were divided into an NF-κB-positive group and an NF-κB-negative group, and the clinical characteristics were compared between the two groups. Kaplan-Meier method was used for survival analysis. Results A total of 414 patients with DLBCL were collected, and EBV-encoded small RNAs were positive in 37 cases (8.9%). Among the 37 EBV-positive patients, NF-κB positivity was observed in 13 cases (35.1%), while among the 377 EBV-negative patients, NF-κB positivity was observed in 144 cases (38.2%), without statistically significant difference (χ2=0.134, P=0.714). No significant difference in clinical characteristics was found between the NF-κB-positive group and the NF-κB-negative group of EBV-positive patients (P>0.05). The 3-year overall survival (OS) rates for the NF-κB-positive and NF-κB-negative groups were 46.2% and 58.3%, respectively, the 5-year OS rates were 19.2% and 29.2%, respectively, and the median OS was 32 vs. 39 months (P=0.441). The corresponding 3-year and 5-year progression-free survival (PFS) rates were 46.2% vs. 41.7% and 19.2% vs. 20.8%, respectively, and the median PFS was 31 vs. 24 months (P=0.933). Conclusion There is no difference in the expression of NF-κB between EBV-positive and EBV-negative DLBCL patients, and the expression of NF-κB has no impact on the clinical characteristics or the prognosis of EBV-positive DLBCL patients.