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The current study was conducted to determine the effects of biofloc systems with different carbon sources and C:N ratios on the growth performance, intestinal morphology and nonspecific immunity response of Nile tilapia (Oreochromis niloticus) fry. The experiment was applied in a 5 × 2 factorial design with 5 different carbon sources and two C:N ratios, comprising 10 treatments for biofloc systems, and a Clearwater system was used as a control. Five different organic carbon sources were tested, in which sugar beet molasses (MO) was progressively replaced with Wheat Flour (WF) including 100% of MO, 75% of MO+ 25% of WF, 50% of MO+ 50% of WF, 25% of MO+ 75% of WF,and 100% of WF. These combinations were applied at two carbon to nitrogen (C:N) ratios, i.e. 15:1 and 20:1. The results showed that, growth performance, water nitrogenous parameters, intestinal villus length and diameter as well as humoral immune responses were improved in the fish cultured in biofloc systems with MO and WF combinations as the organic carbon sources at a C:N ratio of 15. Significant improvements were observed in humoral immune responses (lysozyme activity, total protein, albumin, globulin and hemolytic activity) in all biofloc treatments. The time effect was highly significant for water nitrogenous parameters (TAN, NO 2 and NO 3), meaning that a significant weekly variation existed for all three parameters. Fish in control group had the highest whole body lipid level and lowest protein level compared to the fish treated with biofloc treatments. Finally, Among different biofloc treatment groups, fish in biofloc system with 100% of wheat flour at a C:N ratio of 15:1 showed the highest growth performance with improved intestine histoarchitecture and immune responses. • Growth performance, intestinal morphology and immune responses of O. niloticus improved in biofloc system. • Performance of O. niloticus improved in biofloc system based on higher levels of wheat flour in carbon source composition. • Biofloc in C/N ratio of 15:1 improved water quality and performance of O. niloticus. • Fish reared in bioflock system based on 100% wheat flour and C/N ratio of 15 demonstrated the best performance. [ABSTRACT FROM AUTHOR]