Although traditional mutation is still an attractive approach for strain improvement, it is tedious, time-consuming, and inefficient to screen for surfactin producing strains. To overcome this, we developed a high-throughput screening method for surfactin producing mutants by applying polydiacetylene (PDA) vesicles as sensors with visible chromatic change from blue to red, detected as colorimetric response (CR%) signal, which can even semi-quantify the yields of surfactin. Bacillus subtilis 723 was used as parent strain and multiply mutated with atmospheric and room temperature plasma (ARTP). Mutants were cultured in MicroFlask by Duetz (24 square deepwell plates, Applikon Biotechnology) and surfactin titers were tested in 96-well plates with PDA vesicles. Mutants with surfactin titers above150 mg/L (CR% value above 26%) were selected as high-yield strains and further quantified by HPLC. By integrating MicroFlask cultivation and the PDA vesicles detection, we screened 27,000 mutants and found 37 high-yield strains. From these, one mutant produced 473.6 mg/L surfactin (including 353.1 mg/L C15 surfactin), which was 5.4-fold than that of the parent strain. This method is efficient, cost-effective and provides wider application in screening for various surfactants.