This study was aimed to explore the relation of Treg and invariant natural killer T (iNKT) cell reconstruction with acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. According to the occurrence or absence of aGVHD, 29 pediatric patients who underwent allo-HSCT were firstly divided into two groups non-aGVHD and aGVHD group,then those patients with aGVHD were divided into steroid effective group and steroid resistant group according to their reaction to the steroid treatment. Flow cytometry was used to detect the frequency of Treg cells and iNKT cells in the peripheral blood of the recipients at different time after allo-HSCT(d 15, d 30, d 60, d 90, the time of aGVHD onset and two weeks after steroid treatment). The result showed that the frequencies of Treg cells and the iNKT/T ratio on day 15 in non-aGVHD group were significantly higher than those in the aGVHD group (P < 0.05). It is concluded that a combined monitoring strategy of Treg and iNKT cell reconstruction early after allo-HSCT may facilitate the diagnosis and treatment of aGVHD in children.