Cancer stem cells are thought to be responsible for the post-therapy relapse of primary tumours. Studies have shown that certain cell surface markers are associated with the cancer stem cell phenotype. The AC133 epitope of the CD133 protein has been shown to be a cell surface marker of cancer stem cells in a number of solid tumours, suggesting this could be a suitable target for directed therapy. Targeted therapies, especially monoclonal antibodies have revolutionised the way in which we treat cancers. However, the use of therapeutic antibodies, even humanised antibodies, can lead to detrimental side effects. A novel chemical antibody, known as the aptamer, has emerged as an alternative to conventional antibodies for the targeted treatment of malignant disorders. Here we describe the development of aptamers targeting both the AC133 epitope of the CD133 protein, as well as the CD133 protein. These aptamers are highly specific to AC133 and CD133, as shown through the binding to AC133/CD133 positive cells. They are also efficiently internalised upon cell surface binding, a necessary property required for targeted treatment. Moreover, this aptamer shows superior penetration of tumour spheres, indicating its potential for targeting the core of tumour. This aptamer is currently being functionalised by the addition of a common chemotherapeutic drug, doxorubicin, to determine its effectiveness for the targeted treatment of CD133 positive solid tumours and acute leukaemias.