Abstract: The microbial natural products are important resources for the development of efficient and safe herbicides. In this study, 70 strains of actinomycetes were used as the research object. Firstly, the strains with strong herbicidal activity were screened through the seed germination inhibition test of Arabidopsis thaliana. Then, the herbicidal active substances were tracked, separated and purified by the solvent extraction and various chromatographic techniques with normal phase silica gel, reverse phase silica gel, dextran gel as the media, combined with the activity test. The structure of the compound was analyzed according to the nuclear magnetic resonance spectroscopy data, and finally the herbicidal activity of the pure compound was determined. The results showed that: one strain of Rhodococcus ruber was isolated from 70 strains of actinomycetes, which showed significant inhibitory activity on the seed germination of Arabidopsis thaliana. And a compound designated as compound 1 with herbicidal activity was isolated from the fermentation product of Rhodococcus ruber, which was identified as phenylacetic acid according to NMR data. The herbicidal activity assay showed that the inhibition rate of phenylacetic acid on the seed germination of Arabidopsis thaliana reached 90% at the concentration of 60 µg·mL⁻¹, and the dose test showed that the IC₅₀ value was 36.07 µg·mL⁻¹. The results revealed the application potential of Rhodococcus ruber and its metabolite phenylacetic acid in the development of natural herbicides.