A novel assay of DNA at nanogram levels is presented that is based on the measurement of resonance light scattering (RLS) signals in the presence of norfloxacin. The characteristics of RLS spectra, the effective factors and the optimum conditions of the reaction have been investigated. In Britton-Robinson buffer (pH 5.87), norfloxacin has a maximum peak at 405.5 nm and the RLS intensity is greatly enhanced by trace amounts of DNA due to the interaction between norfloxacin and DNA. Mechanistic studies show that the binding of norfloxacin to DNA forms large particles, which result in the significant enhancement of RLS intensity. The enhanced intensity of RLS is proportional to the concentration of DNA in the range from 0.01–2.0 µg mL−1 for yeast DNA, and from 0.02 to 2.3 µg mL−1 for calf thymus DNA. The determination limit (3σ) is 0.7 ng mL−1 for yeast DNA and 1.2 ng mL−1 for calf thymus DNA, respectively. Synthetic samples were determined satisfactorily.