The interaction of human hemoglobin (Hb) with surface active ionic liquids (SAILs), 1-dodecyl-3-methylimidazolium chloride [C₁₂mim][Cl] and 1-hexyl-3-methylimidazolium dodecylsulfate [C₆mim][SDS], has been studied in aqueous medium through various techniques like surface tension, UV-vis spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The interactional behavior of SAILs toward Hb at the air/solution interface is investigated and various interfacial and thermodynamic parameters have also been calculated. The conformational changes in Hb upon interacting with SAILs have been illustrated from UV-visible measurements in combination with fluorescence spectroscopy. These results indicate that at lower concentration the [C₆mim][SDS] monomer forms stronger Hb–[C₆mim][SDS] monomer complexes as compared to [C₁₂mim][Cl], whereas at higher concentration [C₁₂mim][Cl] denatures Hb more and induces the release of heme from the hydrophobic pocket of Hb. The enthalpy changes were also investigated by using isothermal titration calorimetry (ITC). The dynamic light scattering (DLS) measurements revealed the effect of SAILs on the hydrodynamic diameter (D_h) of Hb.