In this work, the role of room temperature hydrophilic ionic liquids in enhancing the biosensing performance of affinity based electrochemical biosensors in human sweat was investigated. Room temperature ionic liquids (RTILs) consist of an organic cationic and inorganic anionic moieties, with the kosmotropic nature of the anionic moieties providing more stability to the capture probe. The biosensing performance of the immunosensor with capture probe antibody in 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM[BF₄]) and choline dihydrogen phosphate (Choline[DHP]) were compared to capture probe in aqueous buffer. Non-faradaic interfacial charge transfer mechanism was used for evaluating the formation of room temperature ionic liquids (RTILs)/ aqueous human sweat interface. Compact electrical double layers (EDL) are formed by the ionic moieties around the antibodies for enhancing the stability of the antibody capture probes. The stability of the capture probe for biosensing evaluated through zeta potential, indicated the electrostatic repulsion of the RTIL charged moieties encompassing the antibodies, thus preventing any aggregation. The zeta potential measurements also indicated that the antibody in RTIL does not reach the isoelectric point (IEP) even upon interaction with varying pH of sweat. In this study, non-faradaic electrochemical impedance spectroscopy was employed to evaluate the biosensing performance of an affinity based biosensor for the detection of interleukin-6 and cortisol. The EDL interface of the antibody/ionic liquid and sweat buffer interface was modeled using an equivalent circuit for analyzing affinity based biosensing at hybrid electrode/ionic liquid- aqueous sweat buffer interface guided by the choice of the ionic liquid. Interleukin-6 (IL-6) and cortisol that occur in human sweat were detected using the developed affinity based immunoassay. The limit of detection (LOD) of IL-6 in human sweat obtained using capture probe in aqueous phosphate buffered saline was 20 pg/mL. However, a much lower LOD of 0.2pg/mL was obtained in both ionic liquids. Similarly, cortisol of 20 ng/mL was obtained as detection limit using capture probe in aqueous but a better LOD of 0.1 ng/mL was obtained using both the ionic liquids. The better sensing performance in ionic liquids is primarily driven by the enhancement of the double layer capacitance resulting from the formation of compact EDLs. The cross-reactivity studies indicated a better performance of IL-6 detection using Choline[DHP]. On the contrary, an enhanced signal response was obtained for cortisol using cortisol antibody in BMIM[BF₄]. The cross-reactivity studies for cortisol detection using antibody in BMIM[BF₄] did not show any signal response to cross-reactive molecules.