Produced by CD4+ helper T cells and natural killer T (NKT) cells, interleukin-21 (IL-21) performs broad regulatory functions on B cells, CD4+ T cells, CD8+ T cells, NK cells and NKT cells. Targeting IL-21 to enhance the immune system has attracted great interests in the development of vaccination, anti-infection and anti-tumor therapies. Administration of IL-21 in pre-clinical models is however limited by relatively high expense of the recombinant IL-21 protein. Here, we report a rapid and cost-effective method to produce IL-21 using Escherichia coli (E. coli) by introducing a novel two-step dilution strategy for refolding. The method has been validated to produce milligrams of human IL-21, human IL-21/IL-4 chimera and mouse IL-21 with high bioactivities and low endotoxin, mostly suitable for in vitro and in vivo pre-clinical studies.