This study is used to investigate the feasibility of employing the Iodogen method to label triplex-forming oligonucleotide (TFO) targeted to the initiator of the S gene of HBV with 125Ⅰ. A 17-mer oligonucleotides sequence was synthesized and grafted at the 5' terminal with a tyramine group. Radioiodination of the tyramine-TFO with 125Ⅰwas then performed using the Iodogen method. After TFO was labeled with 125Ⅰ using the Iodogen method, the labeling rate, the radiochemical purity, stability and bioactivity were determined, respectively. The results show that the radiolabeling rate and the radiochemical purity were 93% and 99%, respectively; and the radiochemical purity is more than 90% in vitro at -20℃ on the 5th day after labeling; and the rate of 125Ⅰ-tyramine-TFO binding to HepG2.2.15cells was (37.2±1.4) % and statistically different from the rate of HepG2 (p＜0.5). Hence, it is concluded that the labeling of oligonucleotides conjugated with tyramine using the Iodogen method is successful and is characterized with a high labeling rate, high stability, and a low loss of bioactivity of the labeled agent.