Abstract: The effect of glyphosate (GLY) with two surfactants OSA and APG on physiological and biochemical characteristics in Miscanthus floridulus were investigated at a fallow forest land in Kaihua, Zhejiang Province. The results showed that adding surfactants significantly improved the inhibition ratio, but different concentration showed different control ratio. The inhibition ratio of OSA (0.03%) and APG (0.03%) was approximate 80% 86%, the two treatments were higher than the CK by 35.76% and 29.80%, respectively. Adding surfactants significant affected the physiological and biochemical characteristics of M. floridulus. The activity of blade enzyme and TTC root increased from the 3rd day to the 11th day after herbicide treatment (DAT), the concentration of surfactants and activity of antioxidant defense system of M. floridulus significantly enhanced. Antioxidant defense system was imbalanced with increasing of glyphosate and surfactants application time. The content of chlorophyll a, total chlorophyll, and chlorophyll a/b decreased significantly, while the content of chlorophyll b increased significantly. The content of MDA in blade significantly increased and the activity of TTC root decreased on the 29th DAT, the APG (0.03%) had significantly stronger impact on the activity of TTC root than others. It showed that the stability of physiological metabolism and normal function of roots were broken in higher extent after adding surfactants, and accelerated plant wilting and death. M.floridulus showed severely damage symptoms. Therefore, adding surfactant could markedly improve control effect of glyphosate on M.floridulus.