Full spectrum flow cytometry brings a breakthrough for minimal residual disease (MRD) detection in acute myeloid leukemia (AML). We aimed to explore the role of a new panel in MRD detection. We established a 24-color full-spectrum flow cytometry panel. A tube of 24-color antibodies included CD45, CD117, CD34, HLA-DR, CD15, CD64, CD14, CD11c, CD11b, CD13, CD33, CD371, CD7, CD56, CD19, CD4, CD2, CD123, CD200, CD38, CD96, CD71, CD36, and CD9. We discovered that when a tube meets 26 parameters (24 colors), these markers were not only limited to the observation of MRD in AML, but also could be used for fine clustering of bone marrow cells. Mast cells, basophils, myeloid dendritic cells, and plasmacoid dendritic cells were more clearly observed. In addition, immune checkpoint CD96 had the higher expression in CD117+ myeloid naive cells and CD56dimNK cells, while had the lower expression in CD56briNK cells in AML-MRD samples than in normal bone marrow samples. CD200 expression was remarkably enhanced in CD117+ myeloid naive cells, CD4+ T cells, T cells, activated T cells, CD56dimNK cells, and CD56briNK cells in AML-MRD samples. Our results can be used as important basis for auxiliary diagnosis, prognosis judgment, treatment guidance, and immune regulation in AML.