Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system of living organisms and has the ability to reduce the magnitude of stress in humans and animals. In this study, we evaluated the supplemental effects of GABA on normal and high water temperature based on growth, blood plasma composition as well as heat shock proteins and GABA-related gene expression in juvenile olive flounder. For this, a 2 × 2 factorial design of experiment was employed to investigate the dietary effects of GABA at 0 mg/kg of diet (GABA0 diet) and 200 mg/kg of diet (GABA200 diet) in water temperatures of 20 ± 1 °C (normal temperature) and 27 ± 1 °C (high temperature) for 28 days. A total of 180 fish with an average initial weight of 40.1 ± 0.4 g (mean ± SD) were distributed into 12 tanks, of which, each tank contained 15 fish based on the 4 dietary treatment groups in triplicate. At the end of the feeding trial, the results demonstrated that both temperature and GABA had significant effects on the growth performance of the fish. However, fish fed the GABA200 diet had a significantly higher final body weight, weight gain and specific growth rate as well as a significantly lower feed conversion ratio than the fish fed the GABA0 diet at the high water temperature. A significant interactive effect of water temperature and GABA was observed on the growth performance of olive flounder based on the two-way analysis of variance. The plasma GABA levels in fish were increased in a dose-dependent manner at normal or high water temperatures, whereas cortisol and glucose levels were decreased in fish fed GABA-supplemented diets under temperature stress. The GABA-related mRNA expression in the brains of the fish such as GABA type A receptor-associated protein (Gabarap),GABA type B receptor 1 (Gabbr1) and glutamate decarboxylase 1 (Gad1) were not significantly affected by GABA-supplemented diets under normal or temperature stressed conditions. On the other hand, the mRNA expression of heat shock proteins (hsp) in the livers of the fish, such ashsp70andhsp90, were unchanged in fish fed the GABA diets compared to the control diet at the high water temperature. Collectively, the present study showed that dietary supplementation with GABA could enhance growth performance, and improve the feed utilization, plasma biochemical parameters and heat shock proteins and GABA-related gene expression under the stress of high water temperatures in juvenile olive flounder.