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 ¹²⁵I was 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 HepG2.2.15 cells 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.