Precise tumor targeting and therapy is a major trend in cancer treatment. Herein, we designed a tumor acidic microenvironment activatable drug loaded DNA nanostructure, in which, we make a clever use of the sequences of AS1411 and i-motif, which can partially hybridization, and designed a simple while robust DNA d-strand nanostructure, in which, i-motif sequence was designed to regulate the binding ability of the AS1411 aptamer to target tumor, in the normal physiological environment, i-motif inhibits the targeting ability of AS1411. In the acidic tumor microenvironment, i-motif forms a quadruplex conformation and dissociates from AS1411, restoring the targeting ability of AS1411, only when acidic condition and tumor cell receptor are present, this nanostructure can be internalized by the tumor cells. Moreover, the structure change of this nanostructure can realize the release of loaded drug. This drug loaded A-I-Duplex DNA structure showed cancer cell and spheroid targeting and inhibition ability, which is promising in the clinical cancer therapy.