Abstract: Objective To optimize the culture condition of engineered E.coli to improve its expression efficiency of hIL-2-mGM-CSF protein. Methods According to an orthogonal Latin square experiment design, the effects of the culture medium, temperature and IPTG concentration at different levels on the efficiency of the engineered E.coli were evaluated for itsexpression of hIL-2-mGM-CSF protein. The results of SDS-PAGE were analyzed with software and the culture conditions derived from the experimental results were tested in independent cultures. The optimal culture condition was used in three large-scale cultures and the results were compared with that of routine cultures. Results The cultures with TH broth yielded higher relative expression quantity of the target protein than those with 2×YT and LB medium. Compared with the induction temperature at 37℃, induction at 42 ℃ significantly improved the expression efficiency of the target protein. IPTG at the concentration as low as 0.3 mmol/L produced better effect than 1.0 mmol/L IPTG. Statistical analysis suggested that the optimized culture conditions could obviously improve the expression efficiency of the target protein. Large scale cultures with the optimized culture condition resulted in a 5-fold improvement of the relative expression quantity of the protein, which accounted for over 29% of total bacterial protein. Conclusion The optimized culture condition of the engineered E.coli can remarkably increase the expression efficiency of hIL-2-mGM-CSF, which may facilitate the subsequent purification and functional study of the protein.