The effects of 7- and 14-day exposure to water-dissolved Cu at sublethal concentrations 0.01 and 0.1 mg/L and subsequent increase in water temperature at a rate of 8°C/h on intestinal proteolytic activity (PA) and amylolytic activity (AA), as well as brain acetylcholinesterase (AChE) activity and water-soluble protein (WSP) content in juvenile roach Rutilus rutilus have been in vivo studied. Additionally, the values of upper sublethal temperature were determined by the critical thermal maximum (CTM) method. The average values of the CTM (27.5°C) did not differ in fish of control (exposure to 0 μg/L Cu) and Cu-exposed groups. After Cu exposure AA was 22–34% lower, PA was 57–64% lower at Cu concentration 0.01 mg/L, but 27–35% higher at Cu concentration 0.1 mg/L. Subsequent temperature stress did not change the response of proteinases and glycosidases to Cu. An increase in water temperature decreased AA by 21%, AChE activity by 24% in fish of the control group; but increased PA by 32%. AChE activity was 24–28% lower than the control after exposure to Cu (0.1 mg/L and short-term thermal load increased the inhibitory effect of Cu. An increase of WSP content by 36–58% was revealed only after 7 days of exposure to Cu, while temperature stress did not change of the effect. In general, Cu at concentrations found in the aquatic environment can reduce the activity of digestive hydrolases in the intestines of juvenile roach, reducing the rate of assimilation of protein and carbohydrate food components. Inhibition of brain AChE activity is enhanced by subsequent temperature stress. No significant differences were noticed in CTM values in fish of control and Cu-exposed group. The results obtained are important in assessing the environmental risks of chronic exposure to Cu in the zones of thermal pollution of water bodies.