The invention relates to an improved expansion culture medium for regulatory T cells of human cord blood origin and an application method of the expansion culture medium. According to the expansion culture medium, heparin anticoagulated autologous cord blood plasma accounting for 10%-12% of the volume of a culture medium, CD3-CD28 antibody co-expressed immunomagnetic beads, recombinant human interleukin 2, 2-mercaptoethanol, rapamycin, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and gentamicin are added into the RPMI (Roswell Park Memorial Institute)1640 culture medium; and then separated cell suspension is inoculated in a 96-well plates with a U-shaped bottom, hole-division expansion can be performed every 1-2 days, and the expansion period is 3-4 weeks. All reagents in the culture system reach the GMP (good manufacturing practice) level or are originated from autologous cord blood, so that risks caused by ingredients of animal origin are avoided, and the regulatory T cells can be used for a third-party unrelated donor and directly applied to clinical disease treatment; and compared with a traditional culture system, Treg cells (the regulatory T cells) expanded by the improved culture medium is excellent in aspects of growth speed, purity, activity, lymphocyte inhibition function and the like, and the Treg cells are expected to be used as the regulatory T cells of the third-party unrelated donor and applied to the clinical disease treatment.