Monosulfuron was developed for weed control in the field of wheat( Triticum, aestivum L. ) and millet( Panicum miliaceum L. )with the application rate ranging from 15 to 60 g ai/hm2 . Herbicidal activity of monosulfuron was evaluated systematically by bioassay using maize( Zea mays L. ) taproot as indicator and weed fresh weight of Acalypha australis L. and Echinochloa phyllopogon. Maize CAU 3138was the most tolerant cultivars to monosulfuron with IC50 (concentration of 50% inhibition) of 85 μg/kg, Yedan 13 was one of the most sensitive cultivars to monosulfuron with IC50 of 6.4 μg/kg. Monosulfuron inhibited the growth of Acalypha australis L. strongly comparing with that of Echinochloa phyllopogon. Monosulfuron was a good acetolactate synthase(ALS) inhibitor in vitro, the/50 (50% of inhibition) of monosulfuron, chlorsulfuron, tribenuron-methyl and nicosulfuron for CAU 3138 were 32, 2, 19 and 26 nmol/L respectively, for Yedan 13 the /50 were 15, 3, 17 and 65 nmol/L respectively. In vivo ALS inhibition occurred only in higher concentration of 4 sulfonylurea herbicide tested. Comparison study of this test indicated that the mode of action of monosulfuron was the same as that of other sulfonylurea herbicides such as chlorsulfuron, tribenuron-methyl and nicosulfuron, they were all inhibitors targeted at the ALS.