<p><?tk 4?>This study reports the synthesis and comprehensive characterization of a new Schiff-base ligand (L), derived from the condensation of 2,5-thiophenedicarboxaldehyde and sulfamerazine. The structural identity of L was unequivocally confirmed through elemental analysis, FT-IR, ¹H/¹³C NMR spectroscopy, and LC-MS/MS measurements. The electrochemical behavior of L was investigated on a glassy carbon electrode in an anhydrous medium, revealing an irreversible, diffusion-controlled redox process best described by an electrochemical–chemical mechanism. The interaction between L and DNA was examined using voltammetric and spectrophotometric techniques. Differential pulse voltammetry demonstrated a pronounced decrease (44.4%) in the guanine oxidation signal upon interaction with L, indicating strong binding with an estimated binding constant of 1.04 × 10⁶&#xa0;M^–1. Complementary UV–Vis studies showed a hypochromic effect accompanied by a hypsochromic shift in the ligand’s absorption spectrum, consistent with an intercalative binding mode, yielding a Kb value of 1.44 × 10⁶&#xa0;M^–1. Molecular docking simulations supported the experimental findings by revealing that L preferentially binds within the minor groove of DNA. The interaction is stabilized through a network of noncovalent forces, including conventional hydrogen bonds, van der Waals interactions, C–H···X contacts, and π–sulfur contributions, with a calculated binding energy of − 8.8&#xa0;kcal mol^− 1. Overall, the combined electrochemical, spectroscopic, and theoretical results demonstrate that the synthesized Schiff-base ligand L exhibits high affinity toward DNA, positioning it as a promising candidate for further pharmacological evaluation.</p> Graphical abstract