This 10-week study evaluated the potential of cattle manure–derived biochar (produced via slow pyrolysis at 500 ◦C) as a novel carbon source in biofloc systems (C/N ratio 20:1) on water quality, growth, immunity, antioxidant status, microbial activity, and proximate composition of Cyprinus carpio. A total of 1125 fish (8.02 ± 0.85 g) were randomly distributed into 15 tanks (300L) assigned to five treatments: control (Ctr, no carbon), biochar (BC), molasses (M), cattle manure (CM), and a biochar–molasses combination (BC + M). Pyrolysis reduced O/C, H/C ratios and lead, while increasing porosity, pH, C/N ratio (33.68 vs. 11.71), BET surface area (7.24 vs. 0.81 m2/g), mineral oxides (especially those containing P, S, and K), and aromatic C=C stretching. The BC significantly reduced ammonia and nitrite compared to other groups. Final weight was highest in the BC (37.62 vs. 32.29 g). Bacterial counts in water and intestinal fluid were significantly higher in the BC. Leukocyte and erythrocyte counts peaked in the CM, while BC exhibited the lowest levels. The molasses-based group showed the best serum and mucusal immune responses, especially total protein, cortisol, lysozyme, ACH₅₀, respiratory burst activity, leukocyte phagocytosis and antioxidant activities, followed in descending order by BC, CM, BC + M, and the control. BC flocs had highest dry matter and protein, lowest lipids; carcass protein differed only from control, with BC highest in dry matter and control highest in lipid. Overall, cattle manure–derived biochar enhances fish health, productivity, water quality, and sustainability in biofloc system.

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