In drug delivery systems, a stimuli-responsive linker that attaches a targeting carrier and a cytotoxic payload can be dissociated to release the payload on the target over the action of a stimuli, thereby it would harden the selectivity, specificity and potency of the cytotoxic agent against targeted tissues whilst sparing the drug-induced toxicity on normal cells. Oligonucleotide duplexes can unwind and be separated into single-stranded random coils under a defined temperature, and this property makes the oligonucleotide an appealing thermo-responsive linker. In this work, we studied the melting temperatures of different DNA linkers with various lengths and mismatches inserted in the double helix with either different numbers or positions. We further chose the DNA linkers that can unwind at the hyperthermia temperature and used them in the construction of four different drug delivery systems both in vitro and in vivo. Results showed that the chosen DNA linkers in all of the constructed delivery systems can successfully unwind and release cargos or drugs after application of heat compared to control groups. This research demonstrated the potential applications of DNA duplexes as temperature-sensitive linkers of drug delivery systems for cancer therapy.