A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2’-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (d^gC) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2’-OMe-4N-(2-guanidoethyl)-5-methylcytidine (_2’-OMe^gC), 2’-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (_2’-OMe^gC_s), d^gC and 4S-(2-guanidoethyl)-4-thiothymidine (^gsT). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine (^DAN_ac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing ^DAN_ac, d^gC, 2’-OMe-2-thiothymidine (_2’-OMe^sT) and 2’-OMe-8-thioxoadenosine (_2’-OMe^sA) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions.