Bis-naphthalene diimide (1) was successfully synthesized from Fmoc-Lys(Boc)-OH and Fmoc-Lys(NDI)-OH on the automated peptide synthesizer. The absorption spectra of 1 suggested intramolecular stacking of the naphthalene diimide parts in buffer solution. Viscometric titration of a double stranded DNA (dsDNA) solution with 1 suggested that both of the naphthalene diimide parts of 1 are involved in threading intercalation. Kinetic studies suggested that 1 binds to dsDNA with a large affinity constant (K = 106 M(-1)) by "bis-threading intercalation" rather than by electrostatic binding between the cationic sites of lysine parts and phosphate anions of DNA.