Inspired by the biocompatibility of ionic liquids and their dexterousness for the preservation of enzyme structure and activity, herein, the interactions of Cyt-c with naked AuNPs and four IL-mediated AuNPs, which were formed by the fabrication of ILs with common cation 1-ethyl-3-methyl-imidazolium (EMIM) and different anions, to obtain AuNP-IL1 [(BF4)⁻¹ anion], AuNP-IL2 [(CH₃OSO₃)⁻¹ anion], AuNP-IL3 [(CH₃CH₂OSO₃)⁻¹ anion], and (AuNP-IL4) [Cl⁻¹ anion], were studied. Through this work, the peroxidase activity observed in the presence of a lower concentration IL-AuNPs is exceptionally increased (16 fold). IL-AuNPs preferentially counteract the temperature gradient change and long-term solvent preservation effects while persistently maintaining the Cyt-c peroxidase activity without much depreciation. The hydrodynamic diameter (d_H) of the Cyt-c-AuNP system was obtained, which supported the TEM results. Furthermore, to evaluate the effect of Cyt-c interaction with the AuNPs, a Zeta potential analysis was performed. Taken together, the binding of IL-AuNPs with Cyt-c, diameter size analysis, zeta potential, structural integrity evaluation using the DichroWeb software and morphology results suggest the interaction order of the IL-AuNPs to be in a sequence of AuNP-IL2 > AuNP-IL3 > AuNP- IL4 > AuNP-IL1 > Naked AuNPs. Moreover, results indicate that the IL anions play a dominating role in the modulation of interactions between IL-mediated AuNPs and Cyt-c. The study strongly supports the promising character of sulfur-containing IL-mediated AuNPs for Cyt-c immobilization simultaneously opening new avenues for the application of greener and biocompatible nanoparticles with drug delivery and therapeutic applications.