The present work aims to propose a new simple assay for deoxyribonucleic acids (DNA) by applying cationic Gemini surfactant 12-3-12 and cationic dye methylene blue (MB) as resonance light scattering (RLS) probes. The formation of MB-DNA-12-3-12 complex at pH 3.99 results in enhanced RLS signals at 369 nm, which is proportional to the concentration of DNA from 0 to 0.88 mg L−1, with determination limit of 0.014 mg L−1. Most foreign substances do not interfere in the detection and three synthetic samples are analyzed satisfactorily. transmission electron microscopy (TEM), FTIR, and ultraviolet spectra are used to investigate the interaction mechanism.