Objective To establish a human B lymphoma cell line which can stably express Epstein-Barr virus latent membrane protein 1 (LMP1). Methods The LMP1 coding gene with EcoRI and BamHI restriction sites was amplified, cloned into pcDNA3.1-EF1a-mcs-3FLAG-CMV-EGFP plasmid, and positive monoclonal competent cells were picked for PCR and sequencing. The recombinant plasmid was transfected into Ramos cells by electroporation, and the cell line stably expressing LMP1 was picked by G418. The expression of LMP1 in Ramos cells was detected by PCR, Western blot analysis and green fluorescent protein Tag. Results PCR and sequencing confirmed that the LMP1 recombinant plasmid was successfully constructed and a stably transfected Ramos cell line was established. Western blot analysis confirmed that LMP1 protein was successfully expressed in this cell line. Conclusion The Ramos cells stably expressing LMP1 have been successfully established.