Objective To investigate the clinical characteristics of immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. Methods Children with complete clinical data who received allo-HSCT in the department of hematology and oncology from July 2019 to October 2020 were selected as the study subjects. The absolute count of peripheral blood lymphocyte subsets (CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, CD16⁺ CD56⁺ NK cells, CD19⁺ B cells, CD4⁺ CD25⁺ Treg cells) was dynamically monitored on day 0, 14, 21, 28, 60 and 100 after transplantation. The children were divided into non-aGVHD group and aGVHD group according to the presence or absence of acute graft-versus-host disease (aGVHD), and the differences of lymphocyte subsets between the two groups were compared. Results A total of 75 children received allo-HSCT, and 42 of them with complete clinical data after 100 days of transplantation were included in this study. There were 20 boys and 22 girls with a median age of 5.4 (2.8~9.3) years. There were statistically significant differences in the levels of CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, CD16⁺ CD56⁺ NK cells, CD19⁺B cells, CD4⁺CD25⁺ Treg cells and CD4⁺/CD8⁺ ratio at different time points before and after transplantation (P<0.05). CD16⁺CD56⁺ NK cells recovered the earliest and reached the level of pre-transplantation at 21 days post-transplantation. CD3⁺ T cells almost reached the pre-transplantation level at 60 days after transplantation, while CD8⁺ T cells exceeded the pre-transplantation level at 60 days after transplantation. The aGVHD occurred in 16 of 42 children, and the median time of aGVHD occurrence was 20.0 (6.0-36.0) days. At 28d and 60d after transplantation, the absolute count of CD16⁺ CD56⁺ NK cells in aGVHD group was lower than that in non-aGVHD group, the difference was statistically significant (P<0.05). Conclusions CD16⁺ CD56⁺ NK cells were the earliest lymphocyte subsets reconstructed after allo-HSCT in children. NK cell reconstitution is closely related to the occurrence of aGVHD after allo-PBSCT, which may be used as an effective predictor of prognosis in patients with aGVHD.