Nanoparticles have attracted remarkable recent interest for drug delivery application and control of protein structure and activity. The interaction of gold nanoparteicles (GNP), thionine (TH), two complexes of GNP-TH, namely S1 and S2 with the transport protein Human Serum Albumin (HSA) was studied. GNP was prepared by citrate reduction method, while S1 and S2 were synthesized by mixing GNP and TH at different ratios - at room temperature and at 80oC, respectively. GNP, S1 and S2 were characterized by strong plasmon resonance absorption in 500-600 nm region. The adsorption of TH on GNP surfaces was characterized by FTIR spectroscopy. In order to understand the particle size domains of the synthesized GNP, S1 and S2, dynamic light scattering technique was used. Absorbance and fluorescence quenching experiments revealed the formation of strong complexes of S2 and HSA, and comparatively weaker complex between S1 and HSA. Spectroscopic analysis suggested the binding affinity of S1-HSA to be of the order of 104 M−1 and that of GNP-HSA, TH-HSA, S2-HSA to be of the order 105 M−1. Synchronous fluorescence confirmed alteration in the microenvironment of the Trp residues of HSA while practically no shift in the maximum emission wavelength reflected little transformation around Tyr residues. Circular dichroism studies revealed that binding, in all cases, altered the protein conformation by reducing the α-helical content. The binding also caused perturbation of the tertiary structure leading to unfolding of the protein and induced optical activity in the bound molecules, but to varying extents. The present study through multifaceted biophysical experiments is an effort to use GNPs as delivery vehicles for multiple therapeutic purposes.