On the basis of the resonance light scattering (RLS) of Ag nanoparticles (AgNPs), an RLS off-on system was developed for studies of the selective interaction between adriamycin (ADM) and DNA. In this strategy, addition of ADM could induce a proportional decrease in the RLS intensity of AgNPs; this could be used to detect trace amounts of ADM with a detection limit of 12.75 ng mL(-1) in the range 0.021-10.0 μg mL(-1). Subsequently, by investigating the ability of different DNA sequences to restore the RLS intensity of the analytical systems, we found that ADM was selective to dsDNA and had an obvious preference for sequences that were rich in guanine and cytosine bases. In order to validate the results of the RLS assay, fluorescence quenching was used, and binding constants and binding numbers of each system were calculated. Compared with other methods, this RLS off-on strategy was more sensitive, fast, and reliable. It has also supplied a novel method for studying the sequence selectivity of DNA-targeted anticancer drugs and is a novel application of the RLS technique in analytical chemistry.