The invention provides a kit for detecting human regulatory T cell subtypes and a detection method thereof, belonging to the technical field of cell subtype detection. The kit provided by the invention includes the following components: blood dilution solution, mononuclear cell separation solution, cell culture solution, lymphocyte activation solution, dead cell removal dye, FcR blocking agent, fluorescently labeled anti-human cell surface marker molecule Antibodies, fluorescently labeled antibodies against human intracellular molecules, PBS buffer, lipopolysaccharide, washing buffer, cell staining buffer, cell fixative, and permembrane wash. During the detection, the mononuclear cells in the whole blood are first isolated, and then the human peripheral blood mononuclear cells are stimulated, and finally the regulatory T cell subtypes can be detected by fluorescent antibody staining. By using the kit and detection method of the present invention, 2-6 subtypes of regulatory T cells can be easily and quickly distinguished.