Abstract Escherichia coli is widely used to produce recombinant proteins. For soluble overexpression, fusion technology has been developed using different fusion partners. In the present work, we used a newly developed nanoluciferase (NanoLuc) as a novel fusion partner for soluble overexpression of the aggregation-prone interleukin-6 (IL6). Soluble 6×His-NanoLuc-IL6 fusion protein was efficiently overexpressed in E. coli at a yield of approximately 60 mg per liter of culture broth after purification. The fusion partner was removed by enterokinase digestion and monomeric mature IL6 was obtained at a final yield of approximately 15 mg per liter of culture broth. The recombinant IL6 protein was fully active in the receptor activation assay, with a measured EC50 value of 117 ± 10 pM (n = 3). Thus, the novel NanoLuc-fusion approach was efficient for the soluble overexpression of active IL6 protein in E. coli and could be applied to other aggregation-prone proteins in future studies.