Acute lymphoblastic leukemia (ALL) is one of the most common malignant diseases of the hematopoietic system in children. Although the etiology of ALL is unknown, it has been reported that it may be associated with Epstein-Barr virus (EBV) infection. The aim of this study was to analyze the impact of EBV infection on the clinical features and prognosis of childhood ALL. A total of 162 children with ALL admitted to Heilongjiang Provincial Hospital from January 2018 to December 2020 were selected for this stud, and were divided into 2 groups, infected group and non-infected group, according to whether they had EBV infection. Differences in clinical characteristics between the 2 groups were analyzed by χ2 or t-test. The impact of EBV infection on the prognosis of children was analyzed by Kaplan-Meier survival and Cox regression analysis. The 2 groups were statistically significantly different (P<0.05) according to comparison of characteristics such as first symptoms, karyotype, immunophenotyping, clinical risk, whether secondary infection occurred during chemotherapy, and lymphocyte subsets. Logistic regression results suggested that first symptoms, karyotype, immunophenotyping, clinical risk, the presence of secondary infection during chemotherapy, and lymphocyte subsets were independently associated with EBV infection in children with ALL (P<0.05). The complete remission rate at 46 days after chemotherapy, event-free survival (EFS), overall survival (OS), and survival rate were lower in the infected group than non-infected group, and the complete remission recurrence rate was higher than non-infected group (P<0.05). The EBV DNA levels were statistically lower in the good prognosis group (1.07±0.25×103 copies/L) than poor prognosis group (8.86±1.14 ×103 copies/L) (P<0.01). The area under the curve (AUC) for EBV to predict prognosis in children with ALL was 0.921, sensitivity and sensitivity were 86.57%, 80.16%. Infection with EBV is associated with first symptoms, karyotype, immunophenotyping, clinical risk, secondary infection during chemotherapy, and lymphocyte subpopulation index levels in children with ALL, and children with EBV infection have a reduced clinical remission rate and poor prognosis. Therefore, the detection of EBV DNA is clinically important for assessing the prognosis of their disease.