Although DNAzyme is a promising gene therapy agent, low cellular uptake efficiency, poor biological stability, and the unsatisfactory effect of monotherapy limit its development. Herein, a multifunctional DNA nanoassembly (RCA product-aptamer-DNAzyme, RAD) was constructed for cancer cell detection and targeted delivery of doxorubicin (DOX) and DNAzyme. Briefly, the rolling circle amplification (RCA) product was employed as a scaffold, and each repeated sequence was designed to combine with three single-stranded DNA (ssDNA), which carried the aptamer AS1411 sequence, fluorescent group, and DNAzyme sequence, respectively. Up to 40 groups of ssDNA can be assembled into an RCA product, resulting in a high affinity for cancer cells and stronger fluorescent signals. Due to the high binding affinity, RAD displayed high sensitivity for the detection of HepG-2 cells (the limit of detection was 200 cells/mL). In addition, with the formation of the double helix structure, each RAD could load up to 200 DOX molecules. Subsequently, RAD could efficiently and selectively deliver DOX and DNAzyme into cancer cells through the multivalent interaction between the aptamers and membrane nucleolin. Then, the released DNAzyme could recognize and cleave survivin mRNA under the action of Mg2+, leading to the apoptosis of HepG-2 cells for gene therapy, while DOX inserted into intracellular DNA to inhibit cell proliferation, realizing chemotherapy. According to the results, RAD-DOX displayed enhanced therapeutic effects compared with individual gene therapy or chemotherapy, and RAD could protect membrane nucleolin-negative cells from the effects of DOX. Overall, given the enhanced serum stability, high drug-loading capacity, and excellent selective cellular uptake ability of RAD, this strategy shows great potential in the field of cancer therapy.