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An experimental trial was conducted for 90 days to evaluate the growth performance, immunophysiological response of GIFT strain of Tilapia in biofloc‐based rearing system and to assess the relative percentage survival in 3 days after challenging with the virulent strain of Aeromonas hydrophila. Fingerlings with an average body weight 0.98 ± 0.06 g were stocked in triplicate at different stocking densities of 200 (SD1), 250 (SD2), 300 (SD3) and 350 (SD4) m−3 in biofloc‐based treatments and 150 (C) m−3 in control (clear water). Biofloc‐based units (SD1 and SD2) obtained significantly better (P < 0.05) growth performances at the end of the experimental period. Mean body weight of fish in biofloc‐based units showed a decreasing trend with increase in stocking density with 100% survival in all units including control. The stress parameters were significantly lower in biofloc‐based rearing units especially in treatments SD1 and SD2 as compared to the control. The fish from the biofloc‐based units (SD1 and SD2) possessed significantly (P < 0.05) higher immune status as compared to control and other biofloc treatments in terms of respiratory burst, serum lysozyme and myeloperoxidase activity. Relative survival percentages were significantly better in biofloc treatments with highest in SD1 and SD2 (83.33%) after challenge study. GIFT strain of Tilapia at higher stocking densities 200–250 nos m−3 can be taken as optimum stocking density whereas higher stocking densities up to 350 nos m−3 can be reared in the biofloc systems without compromising the growth and immunity.