BackgroundThe interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently availableEscherichia coli-expressed IL-1Ra (E. coliIL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in thepyrGauxotrophAspergillus oryzae.ResultsWe purifiedA. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed thatAsp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life betweenAsp. IL-1Ra andE. coliIL-1Ra.Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life ofAsp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability thanE. coliIL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM).ConclusionThis study reports the production of a functionalAsp.IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed inA. oryzae. Our results suggest thatAsp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative toE. coliIL-1Ra.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12896-023-00785-7.