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In previous study, we identified a novel protein elicitor AMEP412 from Bacillus subtilis BU412, which could trigger plant defense response and induce systemic acquired resistance against diseases. In this paper, the production of the new elicitor was optimised. First, optimal carbon source and nitrogen source were determined as glucose and yeast extract by single factor test. Then, effects of seven different factors on AMEP412 production was studied by Plackett-Burman design. Significant factors (yeast extract, culture temperature and culture time) were selected for further optimisation using central composite design by response surface methodology. The optimum conditions for AMEP412 production were yeast extract 21.72 g/L, culture temperature 30.84 °C and culture time 31.84 h. Under these conditions, the yield of AMEP412 was enhanced from 0.32 to 2.39 mg/mL (7.47-fold), which was quite close to the predicted one. This result significantly raised the yield of AMEP412, which laid solid foundation for its application as a biocontrol agent.