Abstract: In order to improve the production level of aureomycin by Streptomyces aureus and increase the diversity of mutations, the effects of atmospheric pressure room temperature plasma (ARTP), ultraviolet radiation and lithium chloride mutagenesis on the construction of the mutant library of streptomyces aureus were studied. The screening model was established by overcoming the tolerance of precursors and a high concentration of products. Then, after the primary screening and shake flask re-screening, two mutant strains with high yield of aureomycin and stable high potency were selected, of which the colonial morphological characteristics were changed and the aureomycin potency was increased by more than 20% compared with the original strain. Based on the multiple complex mutagenesis and precursor/product tolerance, the screening model of high-yield strains was established, which not only increased the diversity of the mutant library, but also improved the screening efficiency and reduced the screening workload. It had certain application value and guiding significance for reducing the production cost and increasing the production capacity of aureomycin and other antibiotics from streptomyces.