Biotoxicity of selected covalent functionalizaed single-walled carbon nanotubes (SWNTs) was investigated by plasmid DNA cleavage assay. The effect of covalent functionalization on the physical structural and chemical activity of SWNTs was also studied by Vis-NIR spectroscopy, Raman spectroscopy and ROS assay. The DNA cleavage intensity was promoted by covalent functionalization, and the intensity of DNA damage caused by the three types of carbon nanotubes in a descending order was found to be carboxylated SWNTs (SWNT-COOH) > polyethyleneglycol-lated SWNTs (SWNT-PEG) > uncovalent functionalized SWNTs (uSWNTs). SWNTs were efficient intermediate to trigger electron transfer from electron donor to O2 in dark condition which lead to superoxide radical (O2*-) formation. The electron transfer intensity caused by selected SWNTs was also SWNT-COOH > SWNT-PEG > SWNTs. The reason is that defect sites which changed from sp2-hybridized carbon atom on the side wall of carbon nanotubes were more reactive than the perfect ones.