Abstract：Streptomyces lydicus strains A02 is isolated from the soil of suburban vegetable and forest fields in Beijing (China), which is capable of producing natamycin and has proved to be a potential biocontrol agent to several plant fungal diseases. In order to clone the biosynthetic gene clusters and regulate genes of natamycin, the genomic DNA of Streptomyces lydicus A02 is extracted and partially digested with HindIII and BamHI to increase the yield of natamycin by gene modification, the 97-194kb and 48.5-97kb high molecular weight DNA are extracted and ligated to pCC1BAC. The ligation mixtures were transformed into EPI300 competent cells. A total of 800 and 1500 colonies are obtained by white-blue screening. The analysis of enzyme digestion shows that the average size of the insert fragments are about 133kb and 65kb, and the frequency of clones without inserts is less than 1%. The libraries can cover 12.28 times and 11.25 times of genome if the Streptomyces lydicus A02 contains 8Mb chromosome. Therefore, the probability of screening the target fragment from the libraries is more than 99.99%. This BAC library will be an important resource used in gene cloning, the secondary metabolic pathways and new antibiotics.