The purpose of the research is to find out the best method to separate the peripheral blood mononuclear cells (PBMC) from the blood bag, and verify that the mononuclear cells produced by this method can maintain their biological activity after cryopreservation.Firstly, the blood bag was centrifuged with blood bag centrifuge at 2 516g, 4 ℃ for 10 minutes. After obtaining buffy coat, PBMC were separated with blood cell separation solution with 757g for 20 minutes. In this condition, it shows better result of collection. After expansion culture from one-year cryopreservation, more than 95% of effective T cells can be obtained from collected PBMC.The study provides a method to obtain PBMC from blood bags for subsequent use. The advantages of this method are as follows: Cells can be obtained in large quantities at once to reduce individual differences and optimize cell culture process . It can produce different specific immune cells, which can be used as allogeneic immunotherapy in the future. Reduce the waste of biological resources and accelerate the transformation of scientific research resources.