Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiologic agent of koi herpesvirus disease (KHVD) causing morbidity and mortality in common carp and koi L. populations around the world. Recently, knowledge about diagnosis and detection based on nucleic acid has been reported, but validation of serological techniques for virus infection is limited due to lack of effective antibodies. Additionally, fundamental research on the function of structural proteins from CyHV-3 is necessary to understand the relationship between host and virus. ORF136 is one of the predicted envelope proteins incorporated into mature virions. To study the function of proteins encoded by the gene of CyHV-3 and to establish serological methods for detection of CyHV-3, the predicted antigenic determinant based on amino acid sequences of proteins encoded by was amplified using PCR. Then, the recombinant prokaryotic expression vector was constructed by connecting the cloned fragment to prokaryotic expression vector pET-32a (+), used for transformation of Rosetta (DE3) and protein expressions induced by IPTG. New Zealand white rabbits were immunized with the recombinant proteins of pET-32a-ORF136 four times. Furthermore, the antiserums were collected and affinity purification was used to obtain polyclonal antibody after 66 days. Characterization of the polyclonal antibody against the ORF136 protein was further analyzed using western blot and indirect immunofluorescence assays. The expected product size of 381 bp was obtained and the recombinant prokaryotic expression vector pET-32a-ORF136 was confirmed to be constructed as expected using enzyme digestion. As SDS-PAGE analysis indicated that the molecular weight of recombinant protein pET-32a-ORF136 was consistent with the expected size (about 35 kD) and was distributed in the inclusions. Western blot analysis further showed that the purified polyclonal antibody could specifically recognize purified CyHV-3 virions and KS cells infected with CyHV-3. Moreover, immunofluorescence staining showed that specifically green fluorescence was present in the cytoplasm of KS cells infected with CyHV-3, but not in the negative control, further suggesting that CyHV-3 was recognized by the polyclonal antibody. The purified recombinant proteins and effective antibodies are necessary to develop serological methods to detect antigens, except nucleic acid detection method. The purified recombinant proteins could be used as antigens to capture antibodies against CyHV-3 from common carp or koi exposed to KHVD. Similarly, development of a double antibody sandwich ELISA for detection of CyHV-3 also requires an effective polyclonal antibody to capture antigens released from the host. Antibodies produced by recombinant proteins from rabbit or mouse provides the foundation for functional research into structural and nonstructural proteins. In this study, the polyclonal antibody against the ORF136 protein was prepared and analyzed using immunoblotting and immunofluorescence techniques, which provides an essential and valid tool for further research into CyHV-3.