DNA binding investigations are critical for designing betterpharmaceutical compounds since the binding of a compound to dsDNA in theminor groove is critical in drug discovery. Although only one in vitro study onthe DNA binding mode of apigenin (APG) has been conducted, there havebeen no electrochemical and theoretical studies reported. We hereby report themechanism of binding interaction of APG and a new class of sulfonamide-modifiedflavonoids, apigenin disulfonamide (ADSAM) and apigenintrisulfonamide (ATSAM), with deoxyribonucleic acid (DNA). This studywas conducted using multispectroscopic instrumentation techniques, whichinclude UV−vis absorption, thermal denaturation,fluorescence, and Fouriertransform infrared (FTIR) spectroscopy, and electrochemical and viscositymeasurement methods. Also, molecular docking studies were conducted atroom temperature under physiological conditions (pH 7.4). The moleculardocking studies showed that, in all cases, the lowest energy docking poses bind to the minor groove of DNA and the apigenin−DNAcomplex was stabilized by several hydrogen bonds. Also,π−sulfur interactions played a role in the stabilization of the ADSAM−DNA and ATSAM−DNA complexes. The binding affinities of the lowest energy docking pose (schematic diagram of table ofcontent (TOC)) of APG−DNA, ADSAM−DNA, and ATSAM−DNA complexes were found to be−8.2,−8.5, and−8.4 kcal mol−1,respectively. The electrochemical binding constantsKbwere determined to be (1.05×105)±0.04, (0.47×105)±0.02, and (8.13×105)±0.03 for APG, ADSAM, and ATSAM, respectively (all of the tests were run in triplicate and expressed as the mean andstandard deviation (SD)). TheKbconstants calculated for APG, ADSAM, and ATSAM are in harmony for all techniques. As a resultof the incorporation of dimethylsulfamate groups into the APG structure, in the ADSAM−dsDNA and ATSAM−dsDNA complexes,in addition to hydrogen bonds,π−sulfur interactions have also contributed to the stabilization of the ligand−DNA complexes. Thiswork provides new insights that could lead to the development of prospective drugs and vaccines.