Goldfish haematopoietic necrosis virus（GFHNV）is a pathogen causing high mortalities of goldfish，which belongs to Cyprinivirus. In order to establish a high efficient and reliable diagnostic method，a real-time PCR assay for detection of GFHNV was developed in this paper. The primers and TaqMan probes were designed according to the conserved regions of major capsid protein（MCP）gene of GFHNV. And the MCP gene was ligated into the pUC57 plasmid vector to prepare of recombinant plasmid pUC-T/GFHNV as positive control. The detection limit of this Real-time PCR assay was 10 copies of viral gene segment（in pUC-GFHNV），and there was no cross reaction with other 5 aquatic viruses，such as Koi herpesvirus（KHV），Softshell turtle iridovirus（STIV），Epizootic haematopoietic necrosis（EHNV），Channel catfish virus（CCV）and White spot syndrome virus（WSSV）. It's showed that this real-time PCR assay was sensitive and specific for detection of GFHNV and provided an important means for the diagnosis and pathogen detection of the disease.