The invention provides a universal cell processing kit and an application method thereof, and relates to a kit for separating stem cells from human cord blood, bone marrow and peripheral blood and an application method of the kit. The kit contains a reagent A (cell diluent), a reagent B (high-molecular-weight cell precipitant) and a reagent C (medium-density layering agent). The application method of the kit comprises the steps of: firstly, uniformly mixing the cord blood, bone marrow or peripheral blood with the cell diluent, adding the high-molecular-weight cell precipitant, standing for 30 minutes, collecting the supernatant, centrifuging, collecting the concentrated solution, adding the medium-density layering agent to the concentrated solution, centrifuging for 20 minutes, collecting a karyocyte layer and washing with saline for injection 3 times. In the invention, a cell precipitant with a molecular weight of 450000-700000 is used, the survival rate of the separated karyocytes is not lower than 98%, the collection rate of karyocytes is not lower than 85%, and the method provided by the invention is simple, safe and practical, has low cost, can realize industrialized production, and is easy to popularize.