Ammonia is one of the most important aquatic environmental factors, which is of great concern. In order to evaluate the effect of ammonia on guppy (Poecilia reticulate), fish were exposed to increased concentrations (0, 12.50, 25.00, 41.67, 62.50 mg/L) of ammonia for 48 h. After exposure, we measured the anxiety behavior, antioxidant enzymes and pro-inflammation genes (TNF-α, IL-1β and IL-6) of guppy. The results showed that ammonia stress induced fish anxiety, which was manifested by the increased latency to enter the upper half and decreased time spent in upper half compared with control fish. The guppy showed oxidative stress after 48 h of ammonia stress as evidenced by decreases in the activities of antioxidant enzymes and an increase in lipid hydroperoxide content. With prolonged ammonia stress, the expressions of HSP70, HSP90, TNF-α, IL-1β and IL-6 mRNA at first had an increasing trend, and then decreased, all of which were significantly higher than the control levels at 12 h and 24 h after ammonia stress (P < 0.05). Ammonia significantly upregulated these genes mRNA levels after 48 h exposure, suggesting that heat shock proteins and innate immune system may try to protect cells from oxidative stress induced by ammonia stress. Our study showed that higher ammonia exposure induced oxidative stress in exposed fish, since inhibition of antioxidant enzymes activity and increases in lipid peroxidation, and inflammation occurred. Furthermore, the results will be helpful to understand the mechanism of ammonia toxicity in guppys.