Background: A 90-day trial was carried out to investigate the effects of insitu and exsitu biofloc on the immune-physiological responses, nutrient composition and muscle growth-related gene expression. Methods: The experimental groups consisted of IB-1, IB-2, IB-3 (insitu biofloc), EB-1, EB-2, EB-3 (exsitu biofloc). Capacity of 2.4-ton outdoor tanks were stocked with 4.36±0.1 g pearlspot (Etroplus suratensis) juveniles @ 150/m3. Organic jaggery was used as carbon source to maintain 12:1, 15:1, and 20:1 C/N (IB-1, IB-2, IB-3) ratio for insitu biofloc development and exsitu biofloc mass production and feed supplementation at 10, 20 and 30 % graded level in the diet (EB-1, EB-2, EB-3). Result: At the end of the experiment, insitu biofloc system showed significantly better physiological performances in terms of SOD, RBT, MPO, GLU, CAT, and Serum protein compared to exsitu treatment. In exsitu system, biofloc supplemented at 20% significantly improved nutrient composition. There was a significant effect of insitu and exsitu biofloc treatments on whole-body amino acids like Phenylalanine, Leucine, Isoleucine, methionine, tryptophan, and lysine. However, the targeted muscle-growth-related myo D gene was significantly upregulated in IB-3 insitu and EB-2 exsitu treatments, while the myostatin was downregulated in IB-3 and EB-3. Therefore, this present study suggests the adoption of the insitu biofloc method to obtain better performance of Pearlspot, insitu biofloc at 20:1 C: N level could be used to improve overall health and muscle growth in Etroplus suratensis.