Background: Human TNF-related apoptotic-inducing ligand (TRAIL) has been used successfully for targeted therapy of almost all cancers. Leukemia is the most common type of cancer in children, and despite the advances in therapeutic strategies, the survival rate in leukemia cases is very low. Overexpression of interleukin 2 receptor (IL2R) in hematological malignancies has been utilized to target leukemia. Here, we report an immunotoxin fusion construct of human IL2α and TRAIL for targeting leukemia. Aim: Our aim was to develop an immunotoxin to target CD25+ leukemic cells. Methods: Recombinant fusion construct comprising human IL2α and TRAIL114–281 was cloned, expressed and purified. Surface expression levels of IL2α and TRAIL receptors (CD25 and DR5 respectively) were compared in four leukemic cell lines and patient-derived peripheral blood mononuclear cells (PBMCs). Efficacy of immunotoxins was tested in cell lines and PBMCs by cell viability assay and compared with receptor expression. Results: The efficacy of IL2-TRAIL was higher than TRAIL alone and showed an IC50 ranging from 0.2-0.8 μM in cell lines. IL2-TRAIL induced cell death in PBMCs from leukemic patients in vitro, which was proportional to CD25 expression. Out of 34 leukemic samples, 24 samples were susceptible to immunotoxin-mediated cytotoxicity. The efficacy of IL2-TRAIL (87.5 %) was significantly high compared to TRAIL protein (29 %) in both myeloid and lymphoid leukemic patient samples. IL2-TRAIL fusion protein was highly specific for CD25+ leukemia and showed 100 % efficacy in lymphocytic leukemia [acute lymphoblastic leukemia and chronic lymphocytic leukemia] that overexpressed CD25.[Figure not available: see fulltext.] © 2016, Springer International Publishing Switzerland.