The physiological effects of 4 herbicides (butachlor, quinclorac, bensulfuron-methyl and atrazine) on 3 submerged macrophytes (Ceratophyllum demersum, Vallisneria natans and Elodea nuttallii) were tested in laboratory conditions. The variables of the relative growth rate and the photosynthetic pigments content showed that all of the tested herbicides affected the growth of the plants obviously. The abnormal growth of the plants was observed in most treatment groups, even at the lowest concentration (0.0001 mg/L). Except for the C. demersum treated with quinclorac at 0.005 and 0.01 mg/L, the relative growth rates of the plants were inhibited significantly (p < 0.05). Statistical analysis of Chl-a content was carried out with both the t-test and one-way ANOVA to determine the difference between the treatment groups and control. The results showed that Chl-a contents of the plants in all treatment groups were affected by herbicides significantly, except for the C. demersum treated with bensulfuron-methyl at 0.0005 mg/L. The decrease content in Chl-a was positively correlated to the dosage of the herbicides in most treatment groups. It was suggested that herbicides in water body might potentially affect the growth of aquatic macrophytes. Since the Chl-a content of submerged macrophytes responded to the stress of herbicides sensitively and directly, it could be used as biomaker in environmental monitoring or in the ecological risk assessment of herbicide contamination.