Modifying electrode surfaces on the molecule scale allow developing new electrochemical biosensors. A new strategy for the immobilization of calf thymus DNA on the surface of gold nanoparticles which are co-immobilized at a gold electrode through 4,4'-bis(methanethiol) biphenyl (MTP) molecule by assembly process is demonstrated. The DNA modified electrode was incubated in Co(phen)(3)(3+) solution of an aqueous buffer or an acetonitrile (AN) solution, then it was rinsed and placed in a Co(phen)(3)(3+) free buffer solution or AN solution, followed by cyclic voltammetric experiments. Clear redox peaks of Co(phen)(3)(3+) were observed both in an aqueous and AN solutions. The concentration of supporting electrolyte on electrochemical behaviour was discussed. It was found that the surface coverage value of DNA molecules on modified gold nanoparticle and the redox current of adsorbed Co(phen)(3)(3+) were decrease with increasing the size of gold nanoparticles (6, 25, 42, 73, and 93 nm). In aqueous solution, the electron transfer rate constant of Co(phen)(3)(3+/2+) redox couple became slow with increasing the diameter of gold nanoparticle, and the speed almost had nothing to do with the diameter in nonaqueous solution. The surface concentration of Co(phen)(3)(3+) adsorption on DNA modified electrode decreased and rate constant of adsorption kinetics increased with increasing the interactive temperature. In AN solution, the electrostatic interaction between DNA and Co(phen)(3)(3+/2+) was greatly reduced, however, compare with in aqueous solution the interaction between DNA and reduced form of Co(phen)(3)(2+) was more strongly than oxidized form Co(phen)(3)(3+). The surface concentration of Co(phen)(3)(3+) adsorption on DNA modified electrode reach maximum value when the interactive temperature about 20degreesC, and rate constant of adsorption kinetics nearly independent of the interactive temperature. The results show that the DNA can adsorb on the modified electrode firmly and the Co(phen)(3)(3+/2+) adsorbed on DNA give good electrochemical response both in aqueous and nonaqueous solutions. It was confirmed that the DNA modified electrode can be applied in a nonaqueous system and the modified electrode can be used to investigate the interaction between DNA and electroactive species both in aqueous and nonaqueous systems. (C) 2004 Elsevier Ltd. All rights reserved.