Interactions of three cationic gemini surfactants, 12-4-12, 2Br⁻ 12-8-12, 2Br⁻ and 12-4(OH)-12, 2Br⁻ with natured and denatured protein, bovine serumalbumin (BSA) have been studied by means of UV–Visible absorption, steady-state and time-resolved fluorescence, and circular dichromism (CD) spectroscopy. CD spectroscopic study shows the change in the α-helix and β-strand content of protein with the concentration of gemini surfactants. Gemini surfactantwith hydroxyl group in the spacer decreases the α-helix of the BSA more efficiently than that without hydroxyl group in the spacer. Efficiency to decrease the α-helix of the protein increases with decreasing the hydrophobicity of the spacer group of the surfactants at lower concentration range following the order, 12-8-12, 2Br⁻ < 12-4-12, 2Br⁻ < 12-4(OH)-12, 2Br⁻. However, at higher concentration range of surfactant, the increasing order of providing hydrophobic environment to tryptophan (Trp) and tyrosine (Tyr) residues of the protein is as follows: 12-4(OH)-12 < 12-4-12 b 12-8-12. Gemini surfactant with hydrophobic spacer group provides more hydrophobic environment around Trp and Typ residues of the protein forming micelles like structures along the protein chain. In this concentration range, 12-8-12, 2Br⁻ interacts differently as compared to other two surfactants which are evidenced by the data on excited state lifetime of the protein. It is more efficient to form a particular conformer and/or puckerd ring of Trp as compared to other two surfactants. The microenvironment around Trp residues of BSA is perturbed to a greater extent than that around Tyr residues in presence of gemini surfactants. Fluorescence fromTrp and Tyr are quenched by acrylamide to a greater extent in presence of 12-8-12, 2Br⁻. Interactions of denatured BSAwith gemini surfactants also have been studied. 12-8-12, 2Br⁻ even interacts with the BSA unfolded by guanidine hydrochloride (GdHCl) to a greater extent than that by 12-4-12, 2Br⁻ and 12-4(OH)-12, 2Br⁻.