AbstractCyprinid herpesvirus 2 (CyHV‐2) is a viral pathogen worldwide and causing high mortality on goldfish and silver crucian carp (Carassius auratus gibelio). In order to establish a stable and sensitive immunological diagnostic approach, the recombinant ORF121 protein encoded by the CyHV‐2 ORF121 gene, was selected as a capture antigen to identify cells and tissues infected with CyHV‐2 by immunological methods in this study. Firstly, the open reading frame of CyHV‐2 ORF121 was cloned into the PGEX‐4T‐3 vector and expressed in Escherichia coli. Purified recombinant ORF121 protein was then used as an antigen to prepare monoclonal antibodies, and an efficient hybridoma cell line was selected by dot‐blot assay. The resulting mAb‐3D9 was applied to detect CyHV‐2 in infected caudal fin of Carassius auratus gibelio (GiCF) cells and fish tissues by western blotting, immunofluorescence assays and immunohistological asays. The monoclonal antibody could specifically identify CyHV‐2 in infected GiCF cells and the gills, the kidney and the spleen tissues, and it could attenuate CPE by CyHV‐2 in vitro, suggesting it can be applied for CyHV‐2 detection in the crucian carp and ORF121 may be a candidate vaccine against CyHV‐2.