A dual-antibody assay for sero-diagnosis of nasopharygeal carcinoma (NPC) was optimized by evaluating 4 Epstein-Barr virus (EBV) antigen-based enzyme-linked immunosorbent assays (ELISAs).The serum samples of 57 pretreated NPC patients and 58 apparently healthy adults in Guangzhou were collected.The levels of anti-EBV antibody in the sera were tested by 4 ELISAs, which were developed using fusion proteins of glutathione transferase and EBV specific recombinant antigens, namely, EBNA1-IgA, EBNA-IgG, Zta-IgA, and Zta-IgG.When evaluated individually, the sensitivity (0.913) and neg. predictive value (0.9.74) of EBNA1-IA were the highest among the 4 ELISAs tested.Zta-IA test had the highest individual accuracy rate (η, 0.8870) and Youden index (J, 0.7738).The dual positives for EBNA1-IA and Zta-IA were the highest among the 4 dual positives when paired ELISAs were evaluated.Five NPC patients with neg. reaction to EBNA1-IA showed pos. reaction of Zta-IA, and 7 NPC patients with neg. reaction to Zta-IA showed pos. reaction of EBNA1-IA.The EBNA1-IA assay is more suitable than the other 3 ELISAs (EBNA1-IgG, Zta-IA, and Zta-IgG) when used individually for serol. diagnosis of NPC.When two assays are combined, EBNA1-IA and Zta-IA have complementary effect on serol. diagnosis for NPC and it is an optimal combination of serum antibody assays.