Ionic liquids (ILs) are useful in pharmaceutical industries and biotechnology as alternative solvents or sources for protein extraction and purification, preservation of biomolecules and for regulating the catalytic activity of enzymes. However, the binding mechanism, the non‐covalent forces responsible for protein‐IL interactions and dynamics of proteins in IL need to be investigated in depth for the effective use of ILs as alternatives. Herein, we disclose the molecular level understanding of the structural intactness and reactivity of a model protein cytochrome c (Cyt c) in biocompatible threonine‐based ILs with the help of experimental techniques such as isothermal titration calorimetry (ITC), fluorescence spectroscopy, transmission electron microscopy (TEM) as well as molecular docking. Hydrophobic and electrostatic forces are responsible for the structural and conformational integrity of Cyt c in IL. The ITC experiments revealed the Cyt c‐IL binding free energies are in the range of 10–14 kJ/mol and the molecular docking studies demonstrated that ILs interact at the surfaces of Cyt c. The results look promising as the ILs used here are non‐toxic and biocompatible, and thus may find potential applications in structural biology and biotechnology.