Background: Natural killer (NK) cells play a crucial role in immune surveillance and tumor clearance. It has been reported that allogeneic NK cell therapy has good safety and curative effect in patients with refractory and relapsed acute myeloid leukemia (AML). However, the clinical application of adoptive NK infusion is limited due to the lower number of human NK cells. We established a feeder-free NK cell expansion system from peripheral blood (PB) and explore the anti-leukemia potential in preclinical and clinical studies. Methods: The killing capacity of ex vivo-expanded NK cells were detected by Flow cytometry. The in vivo expansion, survival and anti-leukemia activity of the expanded NK cells were detected in K562-bearing NSG mice. Six AML patients with hematological/extra medullary relapsed (n = 3) or persistent positive minimal residual disease (n = 3) after consolidation and maintenance therapy were enrolled in Peking University Institute of Hematology from 2021 to 2022. The patients were treated with high-dose cyclophosphamide and fludarabine, then combined with adoptive infusion of donor-derived expanded NK cells. The chimerism, the number and function of NK cells were analyzed, and the safety and clinical efficacy were evaluated. Results: The ex vivo-expanded NK cells exhibit highly anti-leukemia activity. NK cells proliferate in mice and the CD56⁺ cells to reach the peak time in each organ is 3-7 days and drop to the level before infusion after 14 days. NK cell infusion significantly improve the survival of leukemia mice, and the anti-leukemia effect is NK cell dose-dependent. In the clinical trial, six patients received a total of 7 cases of NK cell infusion, including 3 cases in low-dose group and 4 cases in middle-dose group, among which 1 patient received 2 dose (1 low-dose and 1 middle-dose). In the low-dose group, 1 patient got response (1/3), with the minimal residual disease turned negative. In the middle-dose group, 3 patients responded (3/4), of which 1 patient had the fusion gene CBFB /ABL decreased from 0.99% to 0.13%, 1 case of extramedullary recurrence of AML turned negative, and NK cells could be detected in cerebrospinal fluid, 1 case of hematological recurrence got hematological remission after treatment. The survival time of NK cells in vivo was 7d-30d, with longer time in middle dose group than those in low dose group. The proportion and number of NK cells in responding cases were significantly higher than those in non-responsive group. Conclusions: Clinical grade expanded NK cells has strong anti-leukemia activity in patients with relapse or minimal residual positive AML patients. NK cells can survive for 2 weeks and can enter the central nervous system without obvious adverse reactions. The response rate in middle-dose group is higher than that in low-dose group. However, the efficacy of NK cells still needs to be further confirmed in a large sample size. Clinical trial information: NCT04221971.