Abstract: Ammonia nitrogen accumulation is one of the significant problems in intensive aquaculture, and changes in fish behavior can be used as a sensitive indicator for identifying sudden environmental pollution. In order to study the effects of acute ammonia nitrogen on behavior and physiological characteristics of Lateolabrax maculatus, the present experiment was carried out by using natural seawater as the control group alongside a low-concentration group (5.6 mg/L) and three high-concentration groups (58, 68, and 78 mg/L) with juvenile fish exposed for 6h. The apparent swimming behaviors, tissue structures, and the enzyme activity characteristic were analyzed during the experiment. The results showed that the average swimming speed, acceleration, percentage of movement time, and the nearest neighbor distance of juveniles in the low-concentration group were similar to those in the control group and were relatively stable during the stress process. In contrast, the high-concentration groups showed a decreasing trend as a whole, while the nearest-neighbor distance showed an increasing trend, gradually stabilizing after 4—5h. With the increase of ammonia nitrogen concentration, the average swimming speed, acceleration, and the percentage of movement time of juveniles gradually decreased, while the nearest-neighbor and inter-individual distances gradually increased. Compared with the control group, juveniles in the 5.6 mg/L group began to show congestion of red blood cells in the liver veins and shedding of gill epithelial cells; the necrosis of hepatocytes in juveniles in the 58—78 mg/L group had gradually intensified, and the number of gill lamellae with chloride cells increased, which ultimately led to the serious damage of the tissues and structures. The liver MDA content increased with the increase of ammonia concentration, and the enzyme activities of CAT, SOD, GSH-Px, and LZM firstly increased and then decreased with the increase of ammonia concentration, except for CAT which had the highest activity in the 58 mg/L group, while the other enzyme activities had the highest value in 5.6 mg/L group. The gill Na⁺/K⁺-ATP and Ca²⁺/Mg²⁺-ATP enzyme activities showed an initial increase followed by a decline as ammonia concentration rose, with the highest values found in the 5.6 mg/L group. The results indicate that when ammonia nitrogen concentrations exceed a certain value, the higher the ammonia nitrogen concentration the more serious the damage to the liver and gill tissue structure of juvenile Lateolabrax maculatus will be, which affects the function of the antioxidant and non-specific immune system, and thus the level of its response to ammonia nitrogen stress will gradually decline. This study provides a theoretical basis for developing a healthy aquaculture model for Lateolabrax maculatus by considering both behavioral and physiological aspects.