[Objective] Screened positive strains which had PKSⅠ, PKSⅡ and NRPS genes from 19 endophytic antagonism actinomycetes strains of Sophora alopecuroides L. and preliminary identified types of antibiotics produced by them, which could also provide research methods and theory on rational development and use of endophytic actinomycetes from Sophora alopecuroides L. [Methods] Specific amplified 19 endophytic antagonism actinomycetes by primers of PKSⅠ,PKSⅡ and NRPS genes for screening positive strains. Then 7 kinds of antibiotics were used as contrast and preliminary identified antibiotics produced by positive strains which had PKSⅠ, PKSⅡ and NRPS genes by TLC and HPLC methods. [Results] The rates of positive strains of PKSⅠ, PKSⅡ and NRPS genes were 47.4%, 10.5% and 21.1% respectively. The fermentation broth of 9 endophytic actinomycetes had a peak respectively which had the same elution time with medemycin, the fermentation broth of strain NDZKDS69 had four peaks which had the same elution time with medemycin, acetylspiramycin, teicoplanin and oxytetracycline respectively. [Conclusion] Strepomyces of endophytic actinomycetes from Sophora alopecuroides L. were rich sources of macrolide antibiotic, aromatic polyketide antibiotics and non ribosomal peptide antibiotics. The testing results of molecular fingerprinting and chemical fingerprint chromatography were consistent, and the methods of TLC and HPLC were founded in this paper were convenient, rapid, sensitive and had better repeatability.