To develop an ELISA method using Herpesvirus hominis type 4 (EBV) IgG/Zebra as capture antigen for large population screening. The ELISA method used purified ZEBRA antigen to detect the IgG/ZEBRA antibody from serum in nasopharyngeal carcinoma (NPC) and normal healthy subjects. Of 288 NPC sera, 262 were detected positive, the sensitivity was 91%, while 5 of 96 normal sera were detected positive, the specificity was 94.8% and the results of NPC group and healthy group displayed significant difference (P less than 0.001). IgA/VCA, IgA/EA, IgG/EA in immunoenzyme methods and ZEBRA ELISA were compared during the NPC screening in two cities: Huizhou, Guangdong and Guiping, Guangxi, 5463 and 2017 samples respectively were tested and 5 earlier NPC patients were found. The results indicate that this method has high specificity and sensitivity, and can be used for large population screening to assist early phase NPC diagnosis.