The invention discloses an improved immune fluorescyte staining method, comprising 5 steps: fixing / permeating, permeating, surface staining, staining in nucleus, washing and weight dropping; the method of the invention is an improvement to the Foxp3 immune fluorescyte staining method of eBioscience Company, changes a two-step multiple staining method into a one-step multiple staining method, and carries out the staining on the surface of the cell and in the nucleus simultaneously, thus having the advantages of simple and convenient operation, quick staining, saving reagent, and reducing the cost, etc. The research shows that the method of the invention has no remarkable difference with the method of that of the eBioscience Company, can be used for the staining on the surface of the cell and in the nucleus simultaneously and has extremely good application prospect and promotional value.