Investigation on interaction of azo dyes with bovine serum albumin as carrier protein will be important in the field of toxicology because of distribution and transportation of dyes in blood. In this regard, the interaction between the azo dye, trisodium (4E)-3-oxo-4-[(4- sulfonato-1- naphthyl) hydrazono] naphthalene-2,7-disulfonate (C20H11N2Na3O10S3), known as Amaranth and bovine serum albumin (BSA) was studied using UV-vis absorption, fluorescence spectroscopy, viscosity measurement and molecular docking studies . Also, the association behavior of Amaranth was investigated at its various concentrations and different ionic strength (NaCl) in 5 mM aqueous phosphate buffer of pH 7.0 at 25°C. Spectrophotometric studies of the interaction between Amaranth and BSA have shown that the binding constant was Kb = 7.1×104 M-1 at 298 K. Thermodynamic parameters (ΔH > 0 and ΔS > 0) indicated that hydrophobic forces play major roles in the interaction between Amaranth and BSA. Further, the fluorescent experiments revealed that the quenching mechanism of BSA by Amaranth was static. Also, no obvious increasing of BSA viscosity was observed by addition of Amaranth. The molecular docking method is also employed to understand the interaction of Amaranth with BSA. All these studies confirm that BSA has more affinity towards Amaranth and the groove binding must be predominant.