In order to evaluate the eco-toxicological effects of Roundup® on Carassius auratus (C. auratus), fish were exposed to 32 μg/L Roundup®, isopropylamine salt of glyphosate (G.I.S) and polyoxyethylene amine (POEA) over different periods (0.5, 1, 3, 7 and 14 d). Hydroxyl radical (·OH), malondialdehyde (MDA) and acetylcholinesterase (AChE) in liver were detected in this study. Results showed that the generation of ·OH increased before 7 d, but without significantly difference. ·OH was induced at 1 d for POEA group, 3 d for Roundup® group and 7 d for G.I.S group. At 14 d, ·OH generation returned to normal levels. MDA contents all increased significantly (p ＜ 0.01) during 7 days and then reached a normal level at 14 d. AChE activity in all group tests revealed a significant inhibition (p ＜ 0.01) after 7 days exposure and then rebounded a little, but remained below the control after 14 days exposure. The rate of AChE inhibition range from 13% - 42% in Roundup®, 6% - 40% in G.I.S, and 21% - 54% in POEA, suggesting that POEA was more toxic compared to Roundup® and G.I.S. 32 μg/L Roundup® exposure led to the change of physiological and biochemical indexes in C. auratus, which was a reversible process in the long run.