Abstract: Owing to gradually increased cost in cultivation of Eucalyptus, glyphosate is widely applied for weeding in forest agriculture. However, as non-selected herbicide, glyphosate also severely effect on the growth of Eucalyptus. Therefore, it is important to determine the sensitive concentration and ensure the impact of Eucalyptus to glyphosate, which will provide a basis for breeding transgenic Eucalyptus with resistance to glyphosate. In this study, E. urophylla × grandis clone DH3229 was selected as materials and their growth characters and physiology activity including catalase (CAT), superoxide dismutase(SOD), peroxidase(POD) activities and the contents of malondialdehyde(MDA), proline and chlorophyll were detected under different concentrations of glyphosate spraying. The results showed that spraying glyphosate to plants with diluted 200 times Roundup(2.4g/L glyphosate) could cause severe damages and event to die. The activities of CAT, POD and SOD significantly increased with increasing glyphosate concentration and then decreased. Meanwhile, the activities of CAT and POD reached the highest level under 0.8g/L glyphosate while SOD activity reached the highest level under 1.2g/L glyphosate. However, the contents of MDA and proline continuously increased with increasing glyphosate concentration, suggesting that glyphosate could severely damage the growth of Eucalyptus plant. To further clarify the effect of Eucalyptus growth of glyphosate, their growth and physiology activity of different durations were also determined under 2.4g/L glyphosate spraying. The whole plant die after 16 days’ treatment. The enzyme activities of CAT, SOD and POD increased slowly with the increasing duration and then sharply declined, in which CAT and SOD activities reached the highest after 8d’ treatment and POD activity reached the highest after 6d’s treatment. Chlorophyll content also decreased with the extension of duration. The physiological mechanism of Eucalyptus plants responding to glyphosate stress was analyzed, which provided suitable screening concentration and physiological basis for the cultivation of transgenic Eucalyptus resistant to glyphosate.