The mighty and formidable anti-inflammatory property of recombinant humanized interleukin-1 receptor antagonist (rhIL-1Ra) has been reported against wide range of deadly nightmare cancers and autoimmune diseases, such as rheumatoid arthritis and diabetes mellitus. However, rhIL-1Ra possesses momentary biological half-life (4–6 hours) and is required at high dosages (100-150 mg/day) to augment therapeutic efficacy, resulting in abhorrent toxicity. Therefore, the current study was redeemed with the main objective to egender the engineered rhIL-1Ra Superkine mutant (a “superkine” named as Mutant 1) with superior stability, biological activity and yield. Using PyMOL software, 25 random substitutions were introduced into the wild type rhIL-1Ra protein. The stability of mutants was investigated through molecular dynamic simulation. Mutant-1 (C66S and C122S) was more stable than its wild-type counterpart. The cloning and expression in E. coli BL21 (DE3) utilizing the pET28b vector resulted in the expression of both the wild type and mutant 1 codon-optimized rhIL-1Ra genes. The expressed proteins coupled with his-tagged peptides were purified by a single-step immobilized metal affinity chromatography method with subsequent characterization through SDS–PAGE and western blot analysis. Cysteine-substituted double mutant expression and anti-inflammatory activity was quite comparable and superior to its wild type counterpart. The superkine Mutant 1 (C66S and C122S) reflected seven-fold magnified increase in thymocyte proliferation inhibition activity in contrast to wild type rhIL-1Ra. Cysteine residues have indispensable and decisive role in disulfide bond formation, but the functional role of free cysteine residues in the protein sequence depends upon their position, redox state and the local environment in the protein. In the present study, we engineered a mutant clone of IL-1Ra that expresses higher protein yield with amplified stability and enhanced biological response. This study provides solid rationale to revitalize the robust substitute of wild-type IL-1Ra in clinical and basic research.