The quantitative determination of nucleic acids is of great importance in fundamental research and clinical diagnosis. In this work, the interaction between DNA and cationic Gemini surfactant 12-4-12, which changes the conformation of DNA, was investigated by UV-vis absorption, FT-IR spectra and steady-state fluorescence techniques. A hydrophobic pyrene probe was used to investigate the microenvironment change and calculate the critical micelle concentration (CMC) of Gemini surfactant 12-4-12 (0.69 mmol/L), which is close to the value obtained from the conductivity method (0.79 mmol/L). A new detection assay for DNA is proposed with Gemini surfactant 12-4-12, using the resonance light-scattering (RLS) technique. The formation of DNA12-4-12 complex resulted in enhanced RLS signals at 368 nm, which is proportional to DNA concentration in the range 0.304-5.32 mg/L, with a detection limit of 35 mu g/L. Most coexisting substances do not interfere in the detection and four synthetic samples were analyzed satisfactorily. Copyright (C) 2011 John Wiley & Sons, Ltd.