Abstract: In order to investigate the potential application of the herbicidal active substances in the cottonseed hull, the herbicidal active substances of the cotton husk was extracted by Soxhlet extraction using anhydrous ethanol, n-butanol, petroleum ether and ethyl acetate were selected. The activities of the crude extracts were determined. It was found that anhydrous ethanol was the best solvent for the extraction of the active ingredients from cottonseed hull which could inhibit Echinochloa crusgalli (L.) Beauv.. Based on the gas chromatography-mass spectrography(GC-MS)analysis, 9 main compounds in the ethanol extract of cottonseed hull were detected, including glycerol, tricyclohexyl 3-ene-6-octanone, 4-vinyl-2-methoxyphenol, (o-methylphenol) 2-bromo-2-chloroacetyl ester, myristic acid, ethyl myristate, palmitic acid, isoamyl octanoate and linoleic acid. Further herbicidal activity assays showed that linolenic acid, isoamyl octanoate, 4-vinyl-2-methoxyphenol, and myristic acid were the main components in cottonseed hull extract with certain herbicidal activities. Linoleic acid has the highest inhibitory effect against E. crusgalli, with the IC₅₀ value for the inhibition of the growth of E. crusgalli was 14.5 mg/L.