Abstract: Surfactin is produced from Bacillus subtilis ssp. containing sfp gene (lipopeptide biosurfactants produced by fermentation). Production of surfactin at present is mainly by liquid fermentation, but the production costs are high due to difficulty in purification resulted from addition of the defoaming agent during the production process. Therefore, present invention conducts physical or chemical mutation on Bacillus subtilis subsp. isolated from Thailand seawater shrimp ponds and screens for the mutant strain of Bacillus subtilis subsp. based on sfp gene expression and then produces surfactin from the mutant strain by semi-solid state fermentation.