Epstein-Barr virus (EBV) infects the vast majority of the world’s population. The types of diseases, caused by infection of Epstein-Barr virus, in different subgroups and regions are various. However, the immune mechanism of EBV infection is still unclear. Acute EB virus infection related diseases are usually self-healing. On the other hand, most patients with chronic and latent infections are still lack of effective treatment with a poor prognosis, and risk of cancer. The CRISPR/Cas9 (clustered regular interspaced short palindromic repeats/CRISPR-associated nuclease 9) technology was derived from an acquired immune defense system in bacteria and archaea. As the third-generation genomic editing technology, it can perform targeted editing of specific genomic sequences under the guidance of sgRNA. Because of its characteristics of ease to operation, time saving and high efficiency, it has been widely used in new crop cultivation, animal disease model construction and precise diagnosis and treatment of human diseases. The review describes the progression of CRISPR/Cas9 system in immune research of EB virus infection, including exploration of screening key pathogenic genes and host dependency factors, pathogenic mechanisms, and gene targeted editing to treat EBV-related diseases. It provides implications for studying the pathogenesis of Epstein-Barr virus related diseases and exploring new antiviral treatment strategy.