Objective:To screen anti-EBV-LMP1 Fab of high specificity and affinity from a phage antibody library and identify its binding activity. Methods:Subtractive screening method by alternating SUNE-1/LMP1 cells and solid-phase from a large capacity Fab phagelibrary. After eight rounds screening,30 positive clones were identified by ELISA test. The positive clones were selected by Western blot for Fab soluble expression in TOP 10F’ and the binding activities of Fab with LMP were analyzed in HNE2/LMP1 cell lines. Results:A Fab fragment with fine activity to LMP1 was selected,purified and expressed. The anti-LMP1 Fab binding specificity was confirmed by cell ELISA,FACS,immunofluorescence and immunohistochemistry. Conclusion:The anti-LMP1 Fab binding to nasopharyngeal carcinoma (NPC) cells provides a promising candidate for the biotherapy of EBV-related carcinoma.