Eleutheronema tetradactylum is subordinate to Mugiliformes, Polynemida, Eleutheronema in taxonomy. As a new aquaculture species, it has gained popularity in most farmers in recent years because of the delicious meat, the fast growth, the high market value and other advantages. However, this species is prone to stress from the pulling capture and the transport due to its weak anti-stress ability. In order to understand the effects of acute off-water handling stress on the tissue structure and oxidative stress of juvenile E. tetradactylum, we stocked the juvenile fish in the outdoor water tank and stressed them by simulating the pulling capture. The samples were collected at 2 h, 6 h, 12 h and 24 h after the off-water handling stress, and fixed separately with poly formaldehyde and liquid nitrogen. The samples fixed with poly formaldehyde were used in tissue sectioning and we observed the changes in the micro- structure of gills and muscles. The samples fixed with liquid nitrogen were used to examine the activities of antioxidant enzymes in muscles and the Na+-K+-ATPase in gills. Results showed that varying degrees of damages were induced in the gill lamella, the squamous epithelium cells and mitochondrion-rich cells (MRC) in 2 h after stress. Firstly, the muscle fiber necrosis appeared and then the gap of muscle fiber became widened, followed by vacuolation and even muscle fiber loss. Superoxide dismutase (SOD) and glutathione (GSH) had the same changing pattern, which was a significant drop (P<0.05) in 2 h after stress, followed by the rise to the highest level 12 h later. Then they began to decline slightly lower than the control level at 24 h. Catalase (CAT) and total antioxidant capacity (T-AOC) did not change significantly (P>0.05) at the beginning of 2 h, but then it rose to a highest value at 12 h and subsequently recovered to the normal level at 24 h. The content of malondialdehyde (MDA) did not have a marked change (P>0.05), and it reached the highest value at 6 h, and then fell back to the normal value at 24 h. The activity of Na+-K+-ATPase was elevated (P<0.05) at 2 h, stayed stable, and rose again until 12 h when it reached the highest value, then it went back to the normal value at 24 h. In conclusion, muscles and gills of E. tetradactylum will be damaged by acute off-water handling stress; besides, the damage will deteriorate as the off-water time goes over. The muscle antioxidant enzyme system will only be activated at 2 h after the handling stress. The synergy may exist between SOD and GSH when the organism was stressed by external factors. At 24 h after the stress, the activities of SOD and CAT will be in a state of equilibrium and the values will be significantly lower (P<0.05) than the control level, and this explains that antioxidant enzymes were affected to certain extent by off-water handling stress. In addition, MDA, the degrading product of peroxide lipid, can be used as a biological marker of rapid stress response in E. tetradactylum. Na+-K+-ATPase in gills can provide energy support during this process to improve the anti-stress ability of E. tetradactylum.