Abstract Mitoxantrone (MXT), an anti-tumor antibiotic, shows irreversible electrochemical behavior at a waxed graphite electrode in a 0.05 M Tris–HCl buffer (pH 7.4) solution. The interaction between MXT and calf thymus DNA (ctDNA) in solution has been studied using cyclic voltammetry. An electrochemical equation suitable for examining the binding of irreversibly electroactive molecules to DNA is established. Determination of diffusion coefficients of both free and binding MXT ( D f, D b), the binding constant ( K) and binding site size ( s base pairs per molecule, bp) of MXT with DNA was performed on the basis of the equation. A nonlinear fit analysis of the experimental data yielded: D f=3.76×10 −5 cm 2 s −1, D b=2.73×10 −7 cm 2 s −1, K=8.7×10 9 cm 3 mol −1, s=2.8 bp. The results demonstrate that MXT binds tightly to ctDNA and covers three base pairs. The anthraquinone of MXT, which is a planar heterocyclic ring, intercalates between the DNA's base pairs. The two aminoethylamino side-chains of the drug fit to the major groove reinforce the combination of MXT and DNA. The results show that MXT is a DNA intercalator with a high binding constant compared to those of other anthraquinones.