To establish a quick analytical method using quantitative PCR for marker gene analysis to identify the functions of iTreg cells and subsequently curtail the harvest time for iTreg cells. The data from the marker gene analysis indicated that varying proportions of iTreg cells could reveal the various expression levels of these genes. FoxP3 expression increased to a considerable degree. By using the same iTreg population, the mixed lymphocyte reaction assay was conducted for 5 days. The suppression percentage of T-cells was dependent on the proportion of iTreg cells, indicating that gene expression levels can represent the biological functions of iTreg cells. By using human peripheral blood mononuclear cells for Treg cell induction, the marker gene expression analysis showed a difference between iTreg cells and uninduced T cells. Marker gene analysis requires only 1 day to identify the functions of human iTreg cells can save time in clinical application and might prevent graft-versus-host disease occurrence effectively.