Abstract：Enopeptin A is a depsipeptide with antibacterial, antiviral and a variety of biological activities. The original strain SIIA-H7264 was mutated by UV, NTG, EMS and atmospheric temperature plasma. A high yield and good genetic stability strain A-21 was obtained with a titer of three folds higher than the original strain. The fermentation conditions and fermentation medium were studied by the Plackett-Burmen experiment, the climbing tests and the Box-Behnken method. Under the optimum medium and conditions, the yield of enopeptin A was 1750μg/mL and 2.98 folds higher than that with the initial conditions.