A label-free method for studying DNA sequence recognition of mitoxantrone (MIT) by resonance light-scattering (RLS) technique has been developed in this contribution. Through the RLS spectra, the selective no-covalent interactions between MIT and double-stranded DNA, single-stranded DNA (ssDNA), oligonucleotides were systematically studied. The results of the experiments displayed that MIT had an obvious preference to ssDNA with the K (K(RLS)), 15.16 mmol mg(-1) and the number of binding sites (N), 8.68 × 10(-4) mmol mg(-1). Besides, it was found that MIT had a preference to sequences that were rich in guanine and cytosine bases with K(RLS), 17.29 l mmol(-1) and N, 1.19 × 10(-2) l mmol(-1). The recognition mechanisms were well discussed and by fluorescence method and atom force microscopy, the RLS technique was confirmed to be a reliable method in this study. Compared with other methods, what the RLS strategy displayed was the direct interaction between anticancer drugs and DNA in vitro without the influence of a foreign substance. Thus, it can be a simple, fast and label-free strategy for DNA sequence recognition studies of DNA-targeted anticancer drugs.