DNA-based nanogels have attracted much attention in the biomedical research field. Herein, we report a universal strategy for the fabrication of an aptamer-modified DNA tetrahedron (TET)-based nanogel for combined chemo/gene therapy of multidrug-resistant tumors. In our design, terminal extended antisense oligonucleotides (ASOs) are employed as the linker to co-assemble with two kinds of three-vertex extended TETs for the efficient construction of the DNA-based nanogel. With the incorporation of an active cell-targeting group (aptamer in one vertex of TET) and a controlled-release element (disulfide bridges in the terminals of ASOs), the functional DNA-based nanogel can achieve targeted cellular internalization and stimuli-responsive release of embedded ASOs. After loading with the chemodrug (doxorubicin (DOX), an intercalator of double-stranded DNA), the multifunctional DOX/Nanogel elicits efficient chemo/gene therapy of human MCF-7 breast tumor cells with DOX resistance (MCF-7R). This aptamer-modified DNA tetrahedron-based nanogel provides another strategy for intelligent drug delivery and combined tumor therapy.