Background: This study evaluated growth, body composition, blood biochemistry, antioxidant capacity, innate immunity and ammonia excretion of European grayling (Thymallus thymallus) fed diets containing different protein and lipid contents. Six diets were produced to contain 30, 40 or 50% protein with 10 or 20% lipid and fed to triplicate groups (100 fish per replicate) of fish (25.2 ± 0.28 g) to visual satiety twice daily for 12 weeks. Results: Fish growth was enhanced (P ˂ 0.05) as protein increased from 30 to 40% and plateaued thereafter. Enhancing protein and lipid content of diet led to reduced feed intake and improved feed efficiency. Moreover, protein efficiency ratio increased at higher lipid level while lower values were recorded at higher protein levels. Increasing dietary lipid content led to the enhancement of viscerosomatic index and intraperitoneal fat ratio. An interaction of protein and lipid was found on whole-body lipid, and muscle lipid was responsive to dietary lipid level. Muscle ARA, EPA and Ʃn-6 LC-PUFA contents enhanced by increasing dietary protein level. Moreover, increasing fat content of diet led to enhanced muscle linoleic acid, linolenic acid, ƩMUFA, Ʃn-6, DHA/EPA and n-6/n-3. However, EPA, DHA, Ʃn-6 LC-PUFA, Ʃn-3, Ʃn-3 LC-PUFA and EPA/ARA decreased at higher dietary lipid level. Serum triglyceride (TG) concentration and lactate dehydrogenase (LDH) activity decreased as dietary protein level increased while an opposite effect was observed for cholesterol (CHO) concentration. Increasing fat content of diet led to enhanced serum TG, CHO and glucose concentrations and reduced alanine aminotransferase, aspartate amino transferase and LDH activities. Serum malondialdehyde concentration was enhanced by increasing both dietary protein and lipid contents. Furthermore, serum myeloperoxidase activity was enhanced at higher dietary lipid level. Water total ammonium nitrogen (TAN) concentration was measured after 5 and 24 h of last feeding, and the results indicated the reduction of ammonia excretion as dietary lipid content increased. Conclusions: These findings suggest that 40% dietary protein can support optimal growth of juvenile European grayling and increasing lipid content from 10 to 20% can improve feed utilization and reduce ammonia excretion to the rearing water.