This research aimed to isolate, identify and quantify the predominant allelochemical of yellow sweetclover through organicsolvent extraction, chromatography, thin-layer chromatography (TLC), Gas Chromatography-Mass (GC-MS), and Nuclear Magnetic Resonance (NMR), as well as to evaluate its inhibitory effects on weeds through bioassays. The most active allelochemicals of yellow sweetclover were extracted using petroleum ether. A simple coumarin, identified as 2H-1-benzopyran-2-one, was isolated and recognized as the most active allelochemical, and the chemical structure of this coumarin was determined. The coumarin content of original yellow sweetclover extract was 46.78 μg mL , except for a slight promotion effect on the seed germination of grainamaranth, coumarin exerted significant inhibition effects on both the seed germination and seedling growth of all tested plants (P < 0.05). Coumarin also completely inhibited the seed germination and seedling growth of Italian ryegrass, common knotgrass, and red clover. The coumarin 2H-1-benzopyran-2-one was further found to be the predominant allelochemical of yellow sweetclover. This coumarin had strong inhibition effects on seed germination and seedling growth in many weeds. Therefore, coumarin could be used as a natural herbicide.