Abstract: Streptomyces noursei was rich in secondary metabolites and had a broad-spectrum antifungal activity. A strain of S. noursei NK27, whose secondary metabolites had significant control effect on Botrytis cinerea was screened in our laboratory. In order to improve the ability of NK27 to produce gougerotin, single-factor tests, Plackett-Burman (PB) tests, the steepest climb tests and Box-Behnken tests were designed to optimize the fermentation conditions of NK27. From the five fermentation conditions, the three factors of inoculum age, inoculation amount and the initial pH showed the most significant influence on the yield of gougerotin. The fermentation conditions after the optimization of response surface were as follows: initial pH 7.6, inoculum age 32.6 h, inoculation amount 2.6%, rotation speed 220 r/min, fermentation time 144 h. The content of gougerotin was 3 times higher than that under the original fermentation condition. This study provided a theoretical basis for the industrial fermentation of S. noursei.