A label-free, enzyme-responsive nanosystem that uses a DNA/single-walled carbon nanotube (SWNT) assembly as the substrate is demonstrated for the sensitive, universal detection of restriction and nonrestriction endonucleases as well as methyltransferases in a homogeneous solution on the basis of light scattering (LS) of carbon nanotubes. This protocol is based on the different binding affinities of SWNTs to single-and double-stranded DNA. This difference can lead to different LS signals that can be used for the detection of nuclease cleavage activity. The assay only requires a label-free oligonucleotide probe, significantly reducing the typical cost. The LS technique and the use of a nuclease-specific oligonucleotide probe impart extraordinarily high sensitivity and selectivity. This light scattering assay is universal and label-free with a detection limit of 5 x 10(-6) U mu L-1 for S1 nuclease, 1 x 10(-4) U mu L-1 for EcoRI endonuclease, and 1 x 10(-2) U mu L-1 for EcoRI methylase. In principle, this assay can be used to detect any kind of nuclease by simply changing the DNA sequences of the specific probe.