Recently, much attention has been focused on oligonucleotide drugs that precisely control the gene expression. Among these, triplex-forming oligonucleotides (TFOs) represent common antigene strategies because they bind specifically to the major groove position of genomic DNA to form a triplex DNA structure. Thus far, this promising triplex formation technique represents a successful strategy with strong application prospects for gene manipulation applications (e.g., cancer, Huntington's disease, inflammatory disease, etc.), analytical detection (e.g., nucleic acid, small molecules, etc.), and nanotechnology (e.g., molecular machines, etc.). This review summarizes in detail the full range of potential applications described above, particularly the various chemical modification strategies that have facilitated the stepwise advancement of TFO-based oligonucleotide drugs in recent years to improve the effectiveness, specificity, and applicability of triplex DNA and synergistically promote the effectiveness of triplex DNA.