The invention relates to a human marrow, cord blood or peripheral blood stem cell treating kit and a stem cell separating method. The kit consists of liquid 1, liquid 2 and liquid 3. Based on the prior art, brain protein hydrolysate is added into the liquid 1, so that the stem cells recovery quantity of the treating sample is effectively improved. The kit is used for separating the human marrow from the cord blood or the peripheral blood. Aiming at a cord blood sample, the separated cell recovery sum is improved by 5.9-15.8%, and the separated CD34 stem cell recovery sum is improved by 5.0-8.8%; aiming at a marrow sample, the separated cell recovery sum is improved by 8.7-21%, and the separated CD34 stem cell recovery sum is improved by 8.4-11%; and aiming at a peripheral blood sample, the separated cell recovery sum is improved by 23.1-46.7%, and the separated CD34 stem cells recovery sum is improved by 10.8-25.6%. Due to the improvement of the target stem cells separating and recovering ratio, the human marrow samples, cord blood samples or peripheral blood stem cell samples can be effectively saved, and the use efficiency of the samples can be improved.