High levels of CD25, as part of the IL-2 receptor, are expressed on the surface of the activated T lymphocytes and regulatory T cells, indicating that the soluble CD25 (sCD25) could be a clinically valuable tool for treating several diseases. Moreover, progress has been achieved in targeting the IL-2 receptor to treat autoimmune diseases, organ transplantation and certain hematological malignancies. In the current study, generation of an ssDNA aptamer (Apt51) against CD25 is reported. Apt51 bound to CD25 with high affinity (Kd = 13.4 nM) and specificity. Furthermore, Apt51 was truncated to two shortened variants that almost retained their high affinity for the CD25 protein. Moreover, Apt51 showed good affinity and selectivity for the recognition of CD25 on the cell surface. Importantly, the study showed that Apt51 interfered with the binding of CD25 to its ligand (IL 2) and consequently decreased the IL-2-induced Akt activation.