Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a common subtype of secondary HLH. EBV plays an important part in the course. EBV can cause central nervous system (CNS) infections, and there are few clinical studies on EBV-CNS infection in EBV-HLH patients. All patients who were diagnosed as EBV-HLH and underwent cerebrospinal fluid testing admitted to our center from January 2018 to December 2019 were retrospectively analyzed. Summarized the clinical data, evaluated treatment efficacy after intrathecal injection, and investigated the correlation between EBV-CNS infection with prognosis in EBV-HLH patients. Of 37 of 57 (64.9%) EBV-HLH patients has EBV-CNS infection. The survival of EBV-HLH patients without EBV-CNS infection was significantly better than that in EBV-CNS infection patients (P = 0.018). There were no statistically significant differences in sCD25, ferritin, ALT, AST, LDH, TB, WBC, Hb, and PLT counts between two groups (all P-values > 0.05). Higher EBV-DNA load in peripheral blood was correlated with EBV-CNS infection (P < 0.001). EBV-CNS infection is an independent risk factor affecting the survival of patients (P = 0 .004) . The CSF cell load of patients with and without EBV-CNS infection groups was significantly different (P = 0.024). Intrathecal injection with methotrexate combined with dexamethasone can effectively decrease CSF EBV-DNA load (P = 0.017) and CSF cell load (P = 0.025). EBV-CNS infection is an independent risk factor affecting prognosis in EBV-HLH patients. Therefore, EBV-CNS infection should cause concern for EBV-HLH patients. Cerebrospinal fluid testing is necessary for all patients. Methotrexate combined with dexamethasone intrathecal injection can be an effective treatment for EBV-CNS infection.