The application of the tumor targeting antibody-mediated immunoliposomes (ILP) provides us a potential effective strategy for treating malignancies, such as acute myeloid leukemia (AML). CD123, which is specifically overexpressed on AML cells, plays an important role in cell cycling and enhances the cell resistance to the apoptotic stimuli. Given such a unique role of CD123 in AML cells, we aim to develop a novel drug targeting delivery system using CD123 monoclonal antibody (mAb) in this study. On the basis of the daunorubicin (DNR) loaded PEGylated liposomes (DNR-LP), a post-insertion method was applied to covalently attach the anti-CD123 mAb onto the surface of the liposomes to obtain the anti-CD123 mAb modified immunoliposomes (CD123-ILP). Immunoliposomes with different anti-CD123 mAb density (mAb/liposomal S100PC, molar ratio, 0.06%, L-CD123-ILP and 0.14%, H-CD123-ILP) were prepared, respectively. The expressions of CD123 in KG-1a, Kasumi-1, HL-60, NB4 and THP-1 cells were determined by flow cytometry. The cell binding and uptake assays revealed that CD123-ILP was internalized into the CD123+ AML cells, while the MTT assay indicated that CD123-ILP had stronger inhibitory effect on the growth of THP-1 and KG-1a cells, in which CD123 were highly expressed. Furthermore, in vitro drug release studies of DNR-LP and CD123-ILP showed a sustained release profile for both systems, which were further confirmed by in vivo pharmacokinetics study of liposomal DNR in rats. In this study, we reported the development of CD123-ILP for the first time by our best knowledge, which offered a promising drug targeting delivery system against CD123+ AML cells.