Biomolecular interaction of hydralazine with human serum albumin (HSA) was studied by fluorescence, ultravoilet, three-dimensional, synchronous, Fourier transform infrared, lifetime fluorescence, resonance Rayleigh scattering, circular dichroism, and molecular docking techniques. The intrinsic fluorescence of HSA was quenched by a static quenching mechanism. The effect of beta-cydodextrin on binding was studied. Binding constants and number of binding sites were evaluated using, the Stern-Volmer equation. Thermodynamic parameters (Delta H degrees, Delta G degrees, and Delta S degrees) indicate the involvement of hydrogen bonding with weak van der Waals forces in the interaction. The average binding distance (r) between the HSA and hydralazine was calculated by Fourier resonance energy transfer theory. Molecular docking study confirms the drug binding sites and interaction of hydralazine with amino acid residues.