This study aimed to investigate the association between early immune reconstitution and Epstein-Barr virus (EBV) reactivation by analyzing changes in natural killer (NK), B, and T cells and their functional status in the peripheral blood during the early post-transplant period. This study included 23 patients who underwent haplo-hematopoietic stem cell transplantation (HSCT). The immune reconstitution of NK cells, T cells, and B cells as well as the expression levels of NK and T cell exhaustion markers (PD-1, TIM-3, and CTLA-4) and cytotoxic function at 1, 2, and 3 months post-transplantation were compared between patients with EBV activation (EBV+ group) and those without activation (EBV- group) post- transplantation. EBV activation occurred in nine patients post-transplantation (EBV+ group), whereas 14 patients demonstrated no activation (EBV- group). All patients with EBV activation exhibited EBV viremia, and no EBV-associated diseases occurred. No significant differences in the clinical characteristics were found between the two groups of patients. The median proportion of CD3(+)CD8(+) T cells in the EBV+ group was significantly lower than that in the EBV- group at 1 month post-transplantation (=0.033). The median proportion of the CD3(-)CD16(neg)CD56(bri) subset in the EBV+ group was significantly higher than that in the EBV- group at 2 months post-transplantation (=0.046). No significant differences in the median proportions of CD3(-)CD19(+) B cells were observed between the two groups at 1, 2, and 3 months post-transplantation. The expression of CTLA-4 on CD3(-)CD16(bri)CD56(dim) NK cells in the EBV+ group was significantly higher than that in the EBV- group at 1 month post-transplantation (=0.033). The expression of TIM-3 on CD3(+)CD8(+) T cells in the EBV+ group was significantly higher than that in the EBV- group (=0.009). The expression level of TIM-3 on CD3(-)CD16(neg)CD56(dim) NK cells in the EBV+ group was significantly lower than that in the EBV- group at 2 months post-transplantation (=0.023). The expression levels of TIM-3 on CD3(+)CD4(+) T cells in the EBV+ group than those in the EBV- group at 1 and 3 months post-transplantation (=0.002, =0.043). The median positive rate of Granzyme B expression in CD3(+)CD8(+) T cells and CD3(+)CD4(+) T cells in the EBV+ group was significantly lower than that in the EBV- group at 1-month post-transplantation (=0.033, =0.016). The median positive rate of Granzyme B expression in the CD3(-)CD16(bri)CD56(neg) cell subset in the EBV+ group was higher than that in the EBV- group at 2 months post-transplantation (=0.012). The median positive rate of Granzyme B expression in CD3(+)CD4(+) T cells in the EBV+ group remained significantly lower than that in the EBV- group at 2 months post-transplantation (=0.049). The median positive rate of perforin expression in the CD3(-)CD16(bri)CD56(dim) cell subset was significantly higher in the EBV+ group than in the EBV- group at 3 months post-transplantation (=0.003). The median positive rate of IFN-γ expression in CD3(+)CD8(+) T cells in the EBV+ group was significantly lower than that in the EBV- group at 3 months post-transplantation (=0.036) . Delayed NK cell and T lymphocyte reconstitution, high exhaustion marker expression, and weakened cytotoxic functions may be related to EBV reactivation after haploidentical HSCT.