Nanomaterials employed for enhanced drug delivery and therapeutic effects have been extensively investigated in the past decade. The outcome of current anticancer treatments based on conventional nanoparticles is suboptimal, due to the lack of biocompatibility, the deficient tumor targeting, the limited drug accumulation in the diseased region, etc. Alternatively, DNA-based nanocarriers have emerged as a novel and versatile platform to integrate the advantages of nanotechnologies and biological sciences, which shows great promise in addressing the key issues for biomedical studies. Rather than a genetic information carrier, DNA molecules can work as building blocks to fabricate programmable and bio-functional nanostructures based on Watson Crick base-pairing rules. The DNA-based materials have demonstrated unique properties, such as uniform sizes and shapes, pre-designable and programmable nanostructures, site-specific surface functionality and excellent biocompatibility. These intrigue features allow DNA nanostructures to carry functional moieties to realize precise tumor recognition, customized therapeutic functions and stimuli-responsive drug release, making them highly attractive in many aspects of cancer treatment. In this review, we focus on the recent progress in DNA-based self-assembled materials for the biomedical applications, such as molecular imaging, drug delivery for in vitro or in vivo cancer treatments. We introduce the general strategies and essential requirements for fabricating DNA-based nanocarriers. We summarize the advances of DNA-based nanocarriers according to their functionalities and structural properties for cancer diagnosis and therapy. Finally, we discuss the challenges and future perspectives regarding the detailed in vivo parameters of DNA materials and the design of intelligent DNA nanomedicine for individualized cancer therapy.