A naphthyridine carbamate dimer (NCD) is a synthetic ligand for DNA containing a CGG/CGG sequence. Although NCD can bind selectively and tightly to a CGG/CGG sequence, the highly cooperative 2:1 binding mode has hampered precise analysis of the binding. We describe herein the synthesis of a series of naphthyridine tetramers consisting of two NCD molecules connected with various linkers to seek a ligand that binds to a CGG/CGG sequence exclusively with a 1:1 stoichiometry. Among the tested ligands, NCTB and Z-NCTS, which have linker moieties with restricted conformational flexibility [biphenyl and (Z)-stilbene linker, respectively], gave the exclusive formation of a 1:1 ligand–CGG/CGG complex. The (Z)-stilbene linker in Z-NCTS was designed to have pre-organized conformation appropriate for the binding and, in fact, resulted in the highest binding affinity. Thermodynamic parameters obtained by isothermal titration calorimetry indicated that the stronger binding of Z-NCTS was attributed to its lower entropic cost. The present study provides not only a novel 1:1 binding ligand, but also valuable feedback for subsequent molecular design of DNA and RNA binding ligands.