The invention discloses a method for regulating and controlling synthesis of an extracellular polymeric substance of clostridium acetobutylicum. The method is characterized in that an intron sequence is interpolated into a gene Spo0A of the clostridium acetobutylicum by using a type II intron gene knockout technology. The invention provides a simple and efficient method for regulating and controlling EPS (Extracellular Polymeric Substance) synthesis; a recombinant strain obtained based on the method is relatively good in regulating and controlling effect on the EPS; in case of taking glucose as a carbon source, when cultivated in an anaerobic bottle with the volume of 100 ml by using a slide biomembrane cultivation method, a clostisdium acetobutylicum biomembrane is 5.81 mum in thickness, and the thickness of a strain biomembrane cultivated under the same condition is 24.6 mum.