Abstract: A type of new triplex forming oligonucleotide (TFO) which can form triplex DNA when bound to two similar fragments of homopolypurine/homopolypyrimidine sequences. TFOs were designed according to the above structure to bind the DR region and pre-S gene promoter region of HBV, respectively. The 3′ end of the TFOs can be monophosphorylated or otherwise chemically modified to increase their stability. Cellular experiments prove that these TFOs can be used as drugs to inhibit HBV and treat hepatitis B. TFO and anti-sense oligonucleotide sequences from the DR or pre-S promoter region of HBV can together bind to target RNA sequences and form a (DNA)2:RNA hetero-triplex structure that results in the more efficient inhibition of HBV.