Colloid gold nanoparticle-based layer-by-layer amplification approach was applied to enhance the electrochemical detection sensitivity of DNA hybridization at carbon nanotube modified carbon paste electrodes (CNTPEs). Streptavidin was immobilized onto the surface of CNTPEs, and the conjugation of biotin labeled target oligonucleotides to the above immobilized streptavidin was performed, followed by the hybridization of target oligonucleotides with the gold nanoparticle-labeled DNA probe and then the layer-by-layer enhanced connection of gold nanoparticles, on which oligonucleotides complementary to the DNA probe were attached, to the hybridization system. The differential pulse voltammetry (DPV) signal of total gold nanoparticles was monitored. It was found that the layer-by-layer colloidal gold DPV detection enhanced the sensitivity by about one order of magnitude compared with that of one-layer detection. One-base mismatched DNA and complementary DNA could be distinguished clearly.