AbstractInfections of Cyprinid herpesvirus 2 in goldfish and farmed crucian carp (Carassius auratus gibelio) are still an urgent problem worldwide. Detection and prevention are necessary for the control of haematopoietic necrosis disease caused by CyHV‐2. Although many sensitive molecular diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV‐2 are still important to CyHV‐2 study. In this experiment, purified CyHV‐2 was used as antigens to produce monoclonal antibodies (Mabs). Six Mabs bound to different proteins were selected by Dot‐blot screening and Western‐blot analysis, and no one had cross‐reactivity with closely related koi herpesvirus. Among them, Mabs 2E1‐B10, 1F5‐A3 and 4C4‐A7 belonged to IgG1 isotype, while other three Mabs 3G9‐B11, 3B4‐G5 and 4F4‐B7 belonged to IgM isotype. These six Mabs all could specifically detect CyHV‐2 in CyHV‐2 infected caudal fin of Carassius auratus gibelio (GiCF) cells by immunofluorescence assays. Then, the neutralization ability was tested in vitro, and the result showed that all six Mabs can attenuate CPE by CyHV‐2 in vitro among which 2E1‐B10 had the best neutralization ability. The virus proteins recognized by these six Mabs were identified by mass spectrometry identification, and the result showed they probably were ORF88, ORF55R, ORF115 and ORF151R. This study is the first to prepare Mabs by purifying CyHV‐2, which will provide a practical basis for the in‐depth study of CyHV‐2 virus.