CtIP is one of the key proteins in DNA double strand breaks repair whereby promoting the end resection. It is a known tumor suppressor and interacts with a number of proteins involved in carcinogenesis such as BRCA1, Rb, etc. To better understand the molecular networks of CtIP, CtIP interaction proteins were predicted with online tool PrePPI and PLK1 was found as the novel CtIP interaction protein which plays an essential role in mitosis and also cancer progression. We further validated the novel interaction by immunoprecipitation method. PLK1 showed strong interaction with CtIP. Moreover, Frodock 2.0 tools for docking protein interactions between CtIP and PLK1 was employed. Finally, the immuoprecipitation assay and immunofluorescence staining results showed the interaction between these two proteins are related to DNA damage. Based on these results, we proposed that the CtIP-PLK1 interaction may play important roles in DNA damage response as well as other biological processes.