The invention provides a display device, a display panel and a packaging method of a display substrate, and relates to the technical field of display. The packaging method comprises that: a display substrate is provided, the display substrate comprises a driving backboard and a light-emitting function layer which are arranged in a laminated mode, and the light-emitting function layer comprises a plurality of light-emitting units distributed in an array mode and a pixel definition layer for separating the light-emitting units; at least one packaging layer is formed on the side, deviating from the driving backboard, of the light-emitting function layer, each packaging layer comprises a first inorganic layer, an organic layer and a second inorganic layer, the organic layer is located on the side, deviating from the driving backboard, of the first inorganic layer, and the organic layer is provided with a through hole right opposite to the pixel definition layer; and the second inorganic layer is located on the side, away from the light-emitting function layer, of the organic layer and connected with the first inorganic layer through the through hole. According to the display device, the display panel and the packaging method of the display substrate, stripping of the packaging layer can be avoided, and the packaging effect is improved.