Epstein-Barr virus (EBV) infection occurs commonly in children and presents as a primary or reactivated infection, which are difficult for clinicians to distinguish. This study investigated the clinical characteristics of the two types of infections. Children with detectable plasma EBV-DNA were retrospectively enrolled and divided into primary and reactivated infection group by EBV-specific antibody. We analyzed the patients' characteristics, clinical manifestations, complications, inflammatory biomarkers, and viral load. A total of 9.3% of children with reactivation were immunocompromised over the long-term. The primary infection mostly appeared as infectious mononucleosis (99.8%), while reactivation occurred as an infectious mononucleosis-like disease (65.0%), hemophagocytic syndrome (22.6%), chronic active EBV infection (5.3%) and lymphoma (3.5%). The incidence of fevers, cervical lymphoditis, periorbital edema, pharyngotonsillitis, hepatomegaly and splenomegaly in primary infection were 93.3%, 93.0%, 51.5%, 66.0%, 76.2% and 63.9%, respectively; the incidence of those symptoms in reactivation was 84.0%, 46.9%, 15.4%, 18.5%, 18.5%, and 43.3%, respectively. The incidence of digestive, respiratory, cardiovascular, neurological, hematological, genitourinary complications and multiple serous effusion in primary infection was 68.8%, 18.1%, 8.0%, 0.8%, 2.9%, 0.0% and 2.3%; whereas the incidence of these complications in reactivation was 56.2%, 22.5%, 14.1%, 8.0%, 38.9%, 0.3% and 19.0%. Patients with reactivation were more prone to multi-systemic damage. B-cells were lower, and CD8+ T-cells were higher in primary infection. Viral load was correlated with the level of different cytokines in primary and reactivated infection. EBV primary infection often presents as infectious mononucleosis. The reactivated infection affects more immunocompromised subjects with diverse and complex manifestations. Various complications are more commonly associated with reactivation as a result of different inflammatory responses to different types of infection.