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In order to screen a high-yield spergualin-production strain and an optimal fermentation medium, large numbers of isolates were selected after ultraviolet (UV) mutation and self-tolerant mutation of Bacillus laterosporus A7. A high-yield strain A-94-7 was obtained and the spergualin productivity was 4.1-fold of the parent strain. The genetic stability of the high-yield strain Bacillus laterosporus A-94-7 was very good. After fermentation medium optimization, Bacillus laterosporus A-94-7 was able to produce 380 mg·L-1. The spergualin productivity in Bacillus laterosporus A7 was increased 4.75-fold by mutation and medium optimization.