<p>This 60-day study examined the effects of biofloc technology (C/N ratio of 15:1, using molasses and wheat flour) on water quality, growth, and health in a carp polyculture system, compared to a control group without biofloc. Four carp species, silver carp (<i>Hypophthalmichthys molitrix</i>), common carp (<i>Cyprinus carpio</i>), grass carp (<i>Ctenopharyngodon idella</i>), and bighead carp (<i>Aristichthys nobilis</i>), with initial mean weights of 20, 22, 10, and 10 g, respectively, were stocked in six ponds (8 × 9 × 1 m) at a ratio of 50:25:15:10. Biofloc significantly reduced total ammonia nitrogen (TAN) levels (0.52 vs. 1.05 mg/L) and increased concentrations of nitrate, phosphate, and silica. It also improved final weights of silver and common carp (86.27 and 102.18 g) compared to the control group (71.22 and 87.18 g, respectively). Under biofloc conditions, all species showed significant increases in erythrocyte counts, total serum protein, amylase and lipase activity, total immunoglobulin, myeloperoxidase, and ACH50 levels. Lysozyme activity also increased in silver, bighead, and grass carp. In contrast, leukocyte counts and respiratory burst activity decreased in most species, along with reductions in cortisol and glucose levels across all species. Antioxidant responses varied: catalase activity increased only in bighead carp, while total antioxidant capacity decreased exclusively in grass carp. In conclusion, biofloc technology, likely due to its beneficial microbes (probiotics and heterotrophic ammonia-oxidizing bacteria) and bioactive compounds, improved water quality, growth, and health, especially in silver and common carp, despite increased suspended solids. Overall, combining biofloc with carp polyculture provided synergistic benefits, enhancing fish productivity, water quality, and sustainability.</p>