Background: Epstein-Barr virus (EBV) DNA load monitoring in blood is essential for the diagnosis of EBV-associated diseases. However, the best-suited blood compartment for detection is still under discussion. The aim of this study was to evaluate the diagnostic value of EBV-DNA load in peripheral blood mononuclear cells (PBMC), plasma and whole blood (WB) samples. Methods: A total of 156 patients, including 45 patients with infectious mononucleosis (IM), 57 patients with EBV-associated hemophagocytic lymphohistiocytosis (HLH) and 54 patients with post-transplant lymphoproliferative disorders (PTLD), were enrolled in this study. The EBV-DNA load in PBMC, plasma and WB samples were measured with real-time quantitative polymerase chain reaction (PCR). Results: EBV-DNA load of patients with HLH showed no statistical difference in PBMC, plasma and WB samples, while patients with IM and PTLD showed a higher viral load in PBMC samples. The strongest correlation of EBV-DNA level was found between PBMC and WB samples among patients with IM, HLH and PTLD. The follow-up of EBV-DNA showed that the viral load became negative along with the recovery from the disease, while that in WB and PBMC would remain positive for a long time. Conclusions: For the diagnosis and monitoring of EBV-DNA, the type of specimen should be chosen reasonably according to the disease. As for IM and HLH, plasma is recommended to quantify the EBV-DNA load, while PBMC and plasma are preferred in PTLD.