The invention discloses a method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cells, and relates to a separation, cryopreservation and resuscitation method of adult peripheral blood mononuclear cells. The method comprises the following steps: I, collecting peripheral venous blood of a healthy adult by using a blood taking bag, and separating and purifying the mononuclear cells in the blood; II, adding related cryopreservation reagents necessary for cryopreservating cells so as to cryopreservate the separated mononuclear cells; III, randomly sampling, and detecting bacteria, fungi, endotoxin and mycoplasma in a cell cryopreservation mixed liquid; and IV, resuscitating the cells, suspending in specially prepared cell resuscitation lotion, performing centrifugal washing twice, and further culturing. As the peripheral blood mononuclear cells used in the method are taken from a patient self, the related reagents used in the test are free of exogenous material, related ethical issues can be avoided, and immunological rejection can be avoided after the cells are injected back to the body. The method aims to store healthy adult immune cells for later treatment on diseases related to tumor.