Being a member of WD-40 protein family, retinoblastoma binding protein 4 (RBBP4) plays an important role in histone acetylation, cell cycle progression and differentiation of cancer stem cells. It is important to study new proteins interacting with RBBP4 and to understand its molecular mechanism in the pathogenesis of diseases, including tumors. In this study, the RBBP4 gene CDS sequence was successfully amplified by RT-PCR, and pGBKT7-RBBP4 was constructed by binding with pGBKT7 yeast bait vector. The results of double enzyme digestion and sequencing confirmed that the bait vector of yeast two-hybrid was successfully constructed, which provided a reference for screening new protein interacting with RBBP4 protein by yeast two-hybrid technology.