AbstractInterleukin‐31 (IL‐31), belonging to the IL‐6 cytokine family, is involved in skin inflammation and pruritus, as well as some tumors’ progression. Here, we reported the expression and purification of recombinant human IL‐31 (rhIL‐31) using a prokaryotic system. This recombinant protein was expressed in the form of inclusion bodies, refolded and purified by size‐exclusion chromatography. Circular dichroism analysis revealed that the secondary structure of rhIL‐31 was mainly composed of alpha‐helix, which is in consistence with the 3D model structure built by AlphaFold server. In vitro studies showed that rhIL‐31 exhibited a good binding ability to the recombinant hIL‐31 receptor alpha fused with human Fc fragment (rhIL‐31RA‐hFc) with EC50 value of 16.36 µg/mL in ELISA assay. Meanwhile, flow cytometry demonstrated that rhIL‐31 was able to bind to hIL‐31RA or hOSMRβ expressed on the cell surface, independently. Furthermore, rhIL‐31 could induce the phosphorylation of STAT3 in A549 cells. In conclusion, the prepared rhIL‐31 in this study possesses the binding ability to its receptors, and can activate the signal pathway of JAK/STAT. Thus, it can be applied in further studies, including investigation of hIL‐31‐related diseases, structural analysis, and development of therapeutic drugs, and monoclonal antibodies targeting hIL‐31.