<p>A novel acylhydrazone Schiff base ligand, 2-fluoro-N′-[(1E,2E)-3-(2-methoxyphenyl)prop-2-en-1-ylidene]benzohydrazide (L), was synthesized and coordinated with Ni(II), Cu(II), and Zn(II) ions. The resulting complexes were characterized using TGA–DSC, FTIR/ATR, HRESIMS, ¹H NMR, PXRD, elemental analysis, and complexometric titration, confirming a 1:2 metal-to-ligand stoichiometry and the formation of polycrystalline compounds. Coordination occurs via an N,O-bidentate mode through the enolic oxygen and azomethine nitrogen, forming stable five-membered chelate rings. Thermal analysis revealed high stability, with decomposition leading to the metal oxides, while PXRD indicated crystallite sizes in the 20–50&#xa0;nm range. Spectroscopic DNA-binding studies via UV–Vis and fluorescence spectroscopy demonstrated moderate hypochromism (20–41%) without significant bathochromic shifts, yielding intrinsic binding constants (K_b) on the order of 10⁴&#xa0;L&#xa0;mol⁻¹. Viscosity measurements and ethidium bromide displacement assays corroborated a predominantly non-intercalative groove-binding mode. Molecular docking simulations supported these experimental findings, revealing plausible minor-groove binding poses stabilized by hydrogen bonding, halogen contacts, π–anion interactions, and hydrophobic interactions, consistent with the observed DNA-binding behavior. BSA fluorescence quenching studies suggested a predominant static quenching mechanism, with binding constants in the range of 10⁴–10⁵&#xa0;M⁻¹, indicative of moderate, reversible ground-state complex formation with serum albumin and suggesting relevant protein-binding behavior. The combined experimental and theoretical data demonstrate that metal coordination enhances DNA affinity and modulates biomolecular interactions, particularly for the Ni(II) complex, highlighting the potential of this new acylhydrazone framework as a platform for DNA-binding coordination compounds and related studies of biomolecular interactions.</p>