This work demonstrates an engineering approach towards modulating the physical and the electrochemical properties of Room Temperature Ionic Liquids (RTILs) to facilitate biosensing in affinity based biosensors. RTILs possess wide electrochemical window and high charge storage capacity making them suitable for electrochemical sensors. The interactions between RTILs and biomolecules such as proteins and enzymes are less understood and therefore important in determining the biomolecule stability. RTILs could form a protective layer around protein, retaining their native confirmation there by, enhancing the protein stability as opposed to protein in aqueous buffer, where the interaction of water molecules could cause protein destabilization. This protective layer enhances the overall charge of the protein which is leveraged towards developing electrochemical immuno-sensors. The Hofmeister series is typically used to characterize the stability of proteins, enzymes. We therefore present in this work RTILs such as BMIM[BF₄], Choline[DHP], Choline[TF₂N] based on Hofmeister series and their miscibility (hydrophobicity or hydrophilicity) in aqueous buffers such as human sweat and human serum to determine the stability of an immunoglobulin protein, α-cortisol antibody. Optical characterization techniques such as FTIR and dynamic light scattering (DLS) was used to determine the stability of protein in these chosen RTILs. Since RTILs exhibit excellent charge transfer characteristics, we leverage this towards developing RTIL based point-of care biosensor for the detection of cortisol. The performance of the RTIL biosensor is evaluated using electrochemical techniques such as electrochemical impedance spectroscopy (EIS), chronoamperometry. RTILs contribute to the enhancement of the double layer capacitance upon binding of biomolecules in the electrical double layer (EDL). This modulation of EDL quantifies the performance of the biosensor. Further, even small excitation voltage results in a large change in current response due to excellent electrochemical properties of RTIL. All these characteristics in addition to biomolecule stabilization properties makes RTIL an ideal candidate for electrochemical based biosensing applications.