A 49 day indoor trail with three treatments viz., biofloc (T1), substrate integrated biofloc (T2), substrate (T3) systems and one control (with no biofloc and substrate) in quadruplicate was conducted to evaluate the microbial composition of rearing water, growth performance and immune response of Penaeus vannamei juveniles (4.56 ± 0.13 g) at 10 nos. in 70 L capacity HDPE tanks filled with diluted sea water to 10 g L-1 salinity. Wheat flour was used as carbon source in T1 and T2 at 15:1 C/N ratio. Significantly reduced (P <0.05) total ammonia-N, nitrite and nitrate were observed in treatment tanks than control. The highest total heterotrophic bacteria, Bacillus, Lactobacillus and Vibrio counts were recorded in T2 followed by T1, T3 and lowest in control. Among the experimental groups, T2 recorded with significantly higher (P <0.05) net weight gain (10.38 ± 0.14 g) and lowest food conversion ratio (1.27 ± 0.12) than T1, T3 and control. Survival was significantly lower in control (62.5 ± 0.81%) than treatments. Substrate integrated biofloc (T2) significantly increased (P <0.05) the total haemocyte count (47.24 ± 4.49 x106 cells ml-1), serum protein (82.67 ± 0.01 mg/ml), Phenoloxidase (0.73 ± 0.03, OD 490 nm) and Lysozyme activity (56.32 ± 0.03%) in shrimp compared to T1, T3 and lowest in control. The study revealed that biofloc, substrate and their combination systems have performed significantly better in terms of water quality, beneficial bacterial loads in water, growth and immune response in P. vannamei than the clear water system. Further, it could be concluded that substrate integrated biofloc system resulted in higher production of shrimp.