Four new water-soluble, fluorescent derivatives of dinaphthotetraaza[14]annulene (DNTAA) have been synthesized varying in the structure, dimensions and spatial arrangements of their meso side groups. The products have been carefully characterized by elemental analyses, spectroscopy, crystal structures and quantum-chemical calculations employing DFT methodology. One representative product of the DNTAA series was tested for DNA binding using UV–vis titrations, CD and thermal denaturation experiments. Intercalation of ds-DNA was recognized as a dominant binding mode. In contrast to non-fluorescent phenylene analogues (DBTAA), reported previously, DNTAA derivative showed threefold fluorescence increase upon DNA binding, and offered intriguing new applications as a fluorescent DNA/RNA dye.