Cyprinid herpesvirus 2 (CyHV-2) is an emerging pathogen of the crucian carp, Carassius carassius, which has caused huge economic losses in China and appears to be spreading worldwide. The objective of this study was to produce and characterize new polyclonal antibodies against CyHV-2 ORF121 that were suitable for diagnostic use and the investigation of ORF121 function. The open reading frame (ORF) of 121 was amplified from the culture supernatant of the CyHV-2. ORF121 was subcloned into the pGEX-4T vector and recombinant protein was expressed in Escherichia coli. The purified recombinant protein ORF121 was used to immunize mice and polyclonal antibodies were obtained. The recombinant protein ORF121 was purified under urea conditions then used to 6-week-old BALA/c mice to prepare its polyclonal antibody. Western Blot and indirect fluorescence assay (IFA) assay were used to validate the ORF121 polyclonal antibody. We found the recombinant protein ORF121(rORF121) could be expressed in E. coli. aggregated in the form of inclusion body. The specificity of the anti-ORF121 polyclonal antibody was confirmed by Western Blot. IFA with anti-ORF121 polyclonal antibody for the detection of CyHV-2 infected RyuF-2 cells was developed in this study. In conclusion, the anti-ORF121 polyclonal antibody will be a valuable tool in further studies to elucidate the mechanisms of viral infection and CyHV-2 diagnosis.