A novel electrochemical DNA biosensor system based on multi-walled carbon nanotubes(MWCNTs) was presented in this paper. In order to make multi-walled carbon nanotubes being perpendicularly standing on Au electrode via Au-S chemical bonding, we cut multi-walled carbon nanotubes (1.5 m) into short pipes (200-300 nm). Then, the shortened nanotubes were further modified by thiolization reaction with cysteamine (NH2CH2CH2SH). The –SH modified MWCNTs, gold nanoparticles and 5’ end –SH modified DNA (HS-DNA) as nucleotide probes were self-assembled onto the surface of Au electrode respectively. The DNA biosensors based on self-assembled MWCNTs had a higher current response compared to those based on random MWCNTs. When the target DNA concentration was 4.0×10–9 mol/L to 1.2×10-8 mol/L, the cathodic peak current of Au electrode system with the AQDS as indicator was linearly related, and the detection limit was about 6.5×10-9 mol/L. In addition, the developed DNA biosensors also had a high selectivity of hybridization detection.