This study was carried out to test the effect of acute ammonia stress on antioxidant enzyme activity and mRNA expression levels of HSP70 and HSP90 genes in tissues of Pelteobagrus fulvidraco, and 360 P. fulvidraco [(17.25 ±0.05) g] were randomly exposed to ammonia: 0 (control), 5.70, 28.50 and 57.00 mg/L total ammonia concentrations for 96 h, respectively. Experimental fish were sampled at 0, 12, 24, 48 and 96 h. The results showed that the superoxide dismutase (SOD) activity in liver of fish in low and medium concentration groups increased from 0 to 6 h, and then gradually decreased, but high concentration group continued to decrease. Malondialdehyde (MDA) content in liver of fish in low, medium and high concentration groups was significantly increased throughout the 96-h period; at hour 3, SOD activity in liver of fish in high concentration group was the lowest, while MDA content was the highest. Liver catalase activity increased significantly at hour 24. In low, medium and high concentration groups, the mRNA expression of HSP70 in liver of fish decreased from 0 to 6 h, and then gradually increased, and that in gill of fish continued to increase, but that in brain of fish continued to decreased. The mRNA expression of HSP70 in liver and brain of fish was significantly lower than that of control group at hour 3, but that in gills is opposite. Compared with HSP70, the mRNA expression of HSP90 in liver and gill of fish in high concentration group reached the highest value at hour 24. The results showed that the antioxidant enzymes activity was inhibited by different concentrations of ammonia, and the reason is related to the accumulation of MDA. Compared with HSP90, the mRNA expression of HSP70 in P. fulvidraco was rapidly up-regulated after the occurrence of ammonia stress, and this physiological regulation mechanism suggests that HSP70 plays a more important role in the response to acute ammonia stress.