Surfactant-protein/enzyme interactions have gained widespread interest in modern research due to their implications in various domains like pharmaceuticals, cosmetics, paints, biotechnology, etc. Thus, spectroscopic, microscopic and molecular modeling approaches have been employed to explore the interaction of a novel series of oxy-diester hybrid cationic gemini surfactants, 2,2'-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-alkyl-N,N-dimetliy1-2-oxoethanaminium) dichloride (C-12-E2O-C-12, C-14-E2O-C-14 and C-16-E2O-C-16), with bovine milk xanthine oxidase (XO). Intrinsic fluorescence studies revealed that the concerned gemini surfactants quenched the XO fluorescence through static quenching mechanism. The trend for K-SV values was: C-12-E2O-C-12 C-14-E2O-C-14 > C-16-E2O-C-16). Negative Delta G(b)(0) values confirmed the spontaneity of C-m-E2O-C-m +XO interactions. The results of other techniques (viz., pyrene fluorescence, UV, CD and TEM) also indicated that the concerned gemini surfactants induce conformational changes in XO. Moreover, the actual binding site of C-m-E2O-C-m gemini surfactants into hydrophobic domains of XO was confirmed by molecular docking. This study elucidates the interaction mechanism of high performance gemini surfactants with XO, which in general may be significant to compile the green amphiphilic systems, for industrial and pharmaceutical implications (more specifically to develop, chemical entities for prevention and treatment of various diseases like gout, hyperuicemia, etc.). (C) 2016 Elsevier Ltd. All rights reserved.