Recently, stimuli-responsive DNA nanostructure-based nanodevices have been applied for cancer therapy. In this study, pH-responsive i-motif DNA was modified on gold nanoparticles (AuNPs) via a facile, time-saving freeze-thaw method and utilized to construct stimuli-responsive drug nanocarriers. When the environment pH changes from 7.4 to 5.0, the i-motif DNA would be folded into four-stranded (C-quadruplex) that could be characterized by circular dichroism, and the characteristic of acid stimulate was verified by fluorescence resonance energy transfer (FRET). To enhance specifical cellular uptake, MUC1 aptamer was employed as the targeting moiety. Doxorubicin (Dox) is an anticancer drug that can be efficiently intercalated into GC base pairs of DNA nanostructure to form drug-loaded nanovehicles (Dox@AuNP-MUC1). Additionally, owing to the excellent photothermal conversion efficiency of AuNPs, the synergistic effect between chemotherapy and PTT can be readily achieved by 808 nm near-infrared (NIR) irradiation, which exhibits specifically and efficiently anticancer efficiency. Hence, this multifunctional drug carrier shows the potential for synergistic photothermal-chemotherapy.