[Objective] To improve xiamenmycin yield from Streptomyces xiamenensis 318 we optimized fermentation medium and doubled biosynthetic gene cluster. [Methods] Glucose-yeast extract-maltose medium (GYM) was optimized by a series of single-factor experiments with variation of carbon source (rhamnose and gluconic acid), nitrogen source (KNO3), trace element (ScCl3) and A-Factor analogous (γ-butyrolactone). Subsequently, the biosynthetic gene cluster xim was doubled by site-specific integration to obtain a genetic engineering strain S. xiamenensis 318Pls1 and orthogonal experiment was applied to optimize the medium. [Results] The single-factor experiments improved xiamenmycin yield in wild-type strain 318 from 15 mg/L to 20 mg/L. The yield in biosynthetic gene doubling strain 318Pls1 attained to 35 mg/L in GYM and was increased to 76.15 mg/L in the optimized medium 9# by orthogonal experiment. [Conclusion] This study shows that the medium optimization combined with biosynthetic gene cluster doubling enhanced xiamenmycin yield and provides an opportunity for further improvement of high productivity strain.