The interaction and binding characterization of a sulfa drug, sulfonamide derivative sulfathiazole (Stz) with calf thymus DNA (ctDNA), was investigated using ethidium bromide (EB) as a fluorescence probe by fluorescence spectroscopy, UV–Vis spectroscopy and molecular docking method. The fluorescence quenching mechanism of ctDNA/EB by Stz was static quenching. The UV–Vis absorption spectra, competitive assays with Hoechst 33,258 and ionic strength effect studies showed that the binding of Stz with ctDNA may be minor groove binding mode on the A-T-rich regions of DNA helix. The calculated thermodynamic parameters, ΔH and ΔS, suggested that the driven forces in binding mechanism are hydrogen bonds or van der Waals forces. The molecular docking results were found to be in agreement with the experimental results and confirmed the existence of hydrogen bonds between Stz and DNA.