We prepared Nile Blue (NB)-modified 17-mer DNA duplexes with thiol/disulfide end tethers for immobilization onto gold electrodes to understand the effect of the tether structure of DNA on its electron-transfer (ET) reactions. The longer thiol tether allowed electron transfer between the NB and the electrode through the DNA duplex, whereas the direct ET pathway became predominant in the shorter disulfide tether. Importantly, the insertion of a spacer chain into the disulfide tether changed the direct ET pathway to a through-DNA pathway.