Nasopharyngeal carcinoma (NPC) is a common cancer in Malaysia and elevated serum antibodies to Epstein-Barr virus (EBV) proteins are useful diagnostic markers of the NPC. Coding sequences of EBV proteins LMP2A, EA-D and ZEBRA were cloned from RNA of B95.8 cell line, an EBV-transformed marmoset cell line, into yeast Saccharomyces cerevisiae expression vectors. In this study, ELISA was used to immobilize the recombinant EBV proteins for the detection of serum antibodies in NPC patients. The sensitivities and specificities of serum IgG and IgA against recombinant EBV proteins LMP2A, EA-D and ZEBRA in 124 histopathologically diagnosed NPC and 124 age, gender and ethnic-matched healthy individuals were determined. ZEBRA/IgA was found to be the most sensitive single test, which correctly predicted 90.3% of the NPC cases, followed by LMP2A/IgG (77.4%) and EA-D/IgG (73.4%). For specificity, ZEBRA/IgA, EA-D/IgG and EA-D/IgA were each able to exclude 96.0% of the non-NPC cases. The combination of ZEBRA/IgA and LMP2A/IgG ELISA achieved a sensitivity of 95.2% and a specificity of 99.2%. Among the 124 NPC patients recruited in this study, 100 (80.6%) had elevated VCA/IgA determined by the reference method of indirect immunofluorescence assay (IFA). Thus, a higher sensitivity (95.2%) was achieved by the combination of ZEBRA/IgA and LMP2A/IgG. In addition, the combined ELISA could distinguish the 24 NPC sera which had VCA/IgA titers not detectable by IFA.