[Objective] The hemopoietic necrosis disease of Carassius auratus gibelio infected by Cyprinid herpesvirus 2 (CyHV-2) has caused huge economic losses to the farming industry of this fish. To reveal the mechanism of CyHV-2 infecting host cells might provide an important basis for the investigations of prevention and control technology for this disease.[Methods] In this study, we designed primers for the epitope rich region of CyHV-2 ORF25B, and amplified truncated ORF25B gene by polymerase chain reaction (PCR). Next, we cloned the amplified product into yeast two hybrid bait expression vector pGBKT7. Then the bait recombinant vector pGBKT7-tORF25B was constructed and we transformed it into yeast Y2H Gold. In addition, we detected the transcriptional self-activation and toxicity of the bait protein on the auxotroph medium. Using yeast two hybrid technique, we hybridized the bait strain pGBKT7-tORF25B/Y2H Gold with GiCB cDNA library.[Results] The truncated ORF25B gene was about 981 bp. It was suggested that the bait strain pGBKT7-tORF25B/Y2H Gold was obtained. And the bait vector was shown to have no toxicity to the yeast cells and no self-activation phenomenon to the report genes. There were four candidate host proteins interacting with tORF25B gene coded protein were preliminarily obtained.[Conclusion]] The results of this study have laid a foundation for further study on the protein function of CyHV-2 ORF25B and the mechanism of this virus invasion into host cells.