DNA assembly has provided new opportunities for the development of a novel drug delivery system (DDS) for real-time monitoring and precision treatment of cancer lesions. Herein, we propose mRNA-responsive DNA nanospheres (DNA-NS), whose self-assembly can be triggered by products of rolling circle amplification and functional hairpins and deliver anticancer drug doxorubicin (DOX) for bioimaging and cancer therapy. It has been demonstrated that DNA-NS exhibited good stability in biological environments. Hence, DNA-NS can serve as a universal platform of detections of mRNA related to various tumor cells. DNA-NS can also be applied in the mRNA-dependent DDS. For drug-resistant cells, which are widely present in actual cancer models, DNA-NS can effectively overcome the efflux action of drug-resistant cells to improve the therapeutic efficacy of DOX. In summary, this study provides a potential strategy for constructing the endogenous mRNA-responsive DDS for cancer diagnosis and chemotherapy in vivo.