Aptamer TLS11a was generated using mouse liver cancer cells, but it also shows high binding affinity to human liver cancer cells. Furthermore, TLS11a is the first aptamer to be identified as specific for human liver cancer cells. Our results showed that the target molecule of TLS11a is very likely a membrane protein which can be internalized into cells. These results indicate that TLS11a may be a useful aptamer for targeted drug delivery in liver cancer treatment. Several reports have demonstrated that free Dox is membrane-permeable and can be uptaken by cells through a passive diffusion mechanism, rapidly transported to the nuclei, and bound to the chromosomal DNA. Therefore, while Dox is generally toxic to proliferating cells, it is also toxic to normal cells, thus limiting its therapeutic efficacy in clinical use. Here, by making use of modification of a specific liver cancer aptamer and the intercalation property of Dox, we generated an easy, rapid, and efficient method to deliver Dox to targeted cancer cells. During in vitro experiments, we proved the specific binding affinity of modified TLS11a-GC to target LH86 cells, but non-recognition to human normal liver cells. Furthermore, we demonstrated the specific toxicity of TLS11a-GC-Dox complex to target cells, compared to normal liver cells, thereby limiting its toxicity to only target cells. This targeting was achieved through the specific binding affinity of aptamer TLS11a. Also, less internalization and release of Dox in the TLS11a-GC-Dox group than in the free Dox group was observed using confocal microscopy. Dox itself is a small molecule which can be rapidly uptaken by cells through a passive diffusion mechanism. Within 15 min, cells treated with free Dox show an intense red fluorescence in the nuclear region, indicating that the uptake speed is very rapid. In addition, aptamer internalization required more time than free Dox, thus slowing the internalization of aptamer-Dox complex and the release of Dox from the complex. Therefore, less toxicity was observed in the TLS11a-GC-Dox group than in the free Dox group during in vitro experiments. During in vivo experiments, TLS11a-GC-Dox had decreased cell internalization speed compared to free Dox. However, because of target recognition by TLS11a aptamer, the local concentration of Dox was increased, compared to free Dox. Hence, higher tumor inhibition efficacy was achieved in the TLS11a-GC-Dox-treated mouse group. In summary, by making use of the ability of anthracycline drugs to intercalate between bases of nucleotides, a new design to modify aptamer TLS11a to TLS11a-GC and make Dox and TLS11a-GC conjugates was investigated. The specificity and efficacy of this conjugate to serve as a drug-delivery platform was further demonstrated in vitro and in vivo. Our data showed that the modified aptamer retains its specificity and can load much more Dox than the unmodified aptamer. Also, the aptamer-Dox conjugates are stable in cell culture medium and can differentially target LH86 cells.