Objective：In this study，the conditions for the prokaryotic expression of mouse interleukin-3(IL-3)were screened and optimized，in order to lay the foundation for effective culture of mouse bone marrow -derived mast cells(BMMCs)in vitro. Methods： Nucleotide sequences of mouse IL -3 were optimized according to the commonly used codons in Escherichia coli(E. coli)and it were then synthesized and cloned into the pET -28a(+)vector. The mouse IL -3 plasmid was transformed into E. coli BL21(DE3)and the expression conditions was optimized. The recombinant mouse IL-3 was further purified and applied in the culture of mouse bone marrow -derived mast cells(BMMCs)in vitro to confirm its bioactivity. Results：The plasmid of pET-28a(+)-IL-3 was successfully constructed， and the recombinant mouse IL - 3 was expressed at approximately 16 kDa by SDS - PAGE analysis. The finally purified mouse IL - 3 combined with mouse stem cell factor could induce the differentiation of surface FcεRI and CD117 double positive BMMCs，which further exhibited the function of β-Hexosaminidase release. Conclusion：In this study，the recombinant plasmid for mouse IL -3 was constructed and the expression conditions in E.coli were optimized accordingly. The finally purified mouse IL-3 could be successfully utilized in the culture of BMMCs in vitro for the investigation of allergy.