Objective To establish a reliable assay which can be used clinically to detect the antibody to Epstein-Barr virus-specific DNase (EBV-DNase) for the early diagnosis of nasopharyngeal carcinoma by enzyme-linked immunoelectrotransfer blot technique (EITB). Methods P3HR-1 cells were induced with TPA (12-0-tetradecanoyl-phorbol-13-acetate) to extract EBV-DNase. The sera from 84 patients with nasopharyngeal carcinoma (NPC), 27 patients with other head and neck tumors, and 31 normal healthy adults were detected for EBV-DNase by West Blot and immunochemical staining. Meanwhile,they also underwent EBVCA-IgA examination.Results There were four positive bands in the region between 52 and 59 KD in the sera from NPC patients. The positive rate by EITB for NPC patients was 70.24%, while that for both patients with other tumors and normal adults was 0. The positive rates of EBVCA-IgA were 73.81%, 18.52% and 6.45%, respectively for the three groups.Conclusions EBV-DNase detection with EITB is as sensitive as EBVCA-IgA examination, but with higher specificity, in NPC. It also has the advantage of not using any radioactive material. Therefore, it is a simple and useful method for early diagnosis of NPC.