Interaction of Azobenzene (AB), 4-Hydroxyazobenzene (HAB) and 2,4-dihydroxyazobenzene (or) Sudan Orange G (SOG) with BSA and DNA base (adenine) were investigated by UV-Visible, fluorescence and ATR-IR spectroscopy, cyclic voltammetry and molecular docking methods. With on addition of azo dyes into the BSA and adenine molecules different emission spectral shifts observed, i.e. (i) Upon increasing the concentration of AB molecule, red shifted quenching is noticed in the BSA molecule (338-367 nm), (ii) No significant spectral shifts noticed in the HAB molecule and (iii) Blue shift (338-320 nm) is observed in the SOG molecule. Azo dyes can bind to BSA with stoichiometric ratio of 1:1 and the protein-ligand complexes are stabilized mainly by hydrophobic and van der Waals interaction. IR spectral analysis shows, the azo group (N=N) stretching frequency for AB, HAB and SOG molecules were shifted in the BSA complex. In cyclic voltammetry, the concentration of the dyes added to the BSA solution, a reductive peak appeared in AB, but this peak is not noticed in HAB and SOG. Molecular docking studies specify that the dyes are located within the binding pocket of sub-domain IIIA in BSA and hydrophobic force play a major role in the binding. As a consequence of this study, the addition of a hydroxyl group to the aromatic ring resulted in a decrease in the binding affinity.