Abstract: To radiolabel antisense oligonucleotide (ASON) targeting hTERT mRNA with technetium-99m, and evaluate the characteristics of this novel radiopharmaceutical. One 18mer antisense uniformly phosphorothioate-modified oligonucleotide targeting hTERT mRNA was radiolabeled with technetium-99m through bifunctional chelator N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (S-Acetyl NHS-MAG₃). The novel molecular imaging agent was characterized in vitro. All data were analyzed by the statistic software SPSS12.0. The labeling efficiencies of ⁹⁹Tc^m-hTERT ASON reach 76%±5% ( n =5) within 15 min to 30 min at room temperature, the specific activity is up to 1850 GBq/g, and the radiochemical purity is above 96% after purification. Control labeling of ASON without bifunctional chelator under the same conditions shows only about 5.0%±1.6% ( n =5) binding, which is significantly different compared with the results of labeling of ASON through bifunctional chelator ( P <0.05). The radiochemical purity of ⁹⁹Tc^m-hTERT ASON at room temperature, as well as that of in normal saline at room temperature and in 37 ℃ fresh human serum, always remains above 93% periodically over 24 h. The result of PAGE analysis indicates the non-degradation of ⁹⁹Tc^m-hTERT ASON by the nuclease in serum after its incubation in 37℃ fresh human serum for 1, 2, 4 and 6 h respectively. After incubated in 37 ℃ fresh human serum for 1, 3 and 6 h respectively, the protein-binding ratio of ⁹⁹Tc^m-hTERT ASON are 15.0%±1.6%, 18.0%±1.9%, and 20.0%±2.4%. hTERT ASON can be radiolabeled with technetium-99m through bifunctional chelator successfully. The antisense molecular imaging probe maintains good stability and low protein-binding, which may be a suitable antisense probe for the further antisense imaging study.