Telomerase presents over-expression in most of cancer cells and has been a near-universal marker of cancer. Studies revealed that inhibiting telomerase activity by utilizing oligonucleotides to down-regulate the expression of intracellular human telomerase reverse transcriptase (hTERT) mRNA was an effective method to achieve anti-tumor therapy. Considering oncogenic microRNA-21 has been proved to indirectly up-regulate hTERT expression and drive cancer metastasis and aggression through increased telomerase activity, here we constructed a AS1411-functionallized oligonucleotide-conjugated gold nanoprobe (Au-nanoprobe) to simultaneously down-regulate intracellular microRNA-21 and hTERT mRNA by using anti-sense oligonucleotide technology to explore their targeted anti-tumor therapy effect. In vitro cell studies demonstrated that Au-nanoprobes could effectively induce apoptosis and inhibit the proliferation of cancer cells through down-regulating intracellular hTERT activity. In vivo imaging and anti-tumor studies revealed that Au-nanoprobes could accumulate at the tumor site and inhibit the growth of MCF-7 tumor xenografted on balb/c nude mice, thus exert the potential of anti-tumor therapy.