Abstract: The effects of different densities of Bacillus substilis on water quality and on the immunity indicators of larval Penaeus vannamei were studied. The results demonstrated that B. substilis could significantly (P < 0.05) decrease the contents of COD, NH₃-N and inhibit the production of NO₂-N. With a density of 1.25×10⁴ cfu·mL^-1 of B.substilis, the average contents of COD, NH₃-N and NO₂-N of the treated groups decreased 65.30%, 59.70% and 88.64%, respectively. After 20-day cultivation, alkaline phosphatase (AKP), peroxidase (POD), phenoloxidase (PO), superoxide dismutase (SOD), the activity of anti-bacterial enzyme and bacteriolysis of Penaeus vannamei increased 3.67, 1.64, 0.45, 3.06, 2.57 and 1.14 times, respectively. Moreover, the survival rate of the treatment groups increased 10% and the weight gain increased 2.44 times when compared with the control group. Therefore, it showed that appropriate density of B.substilis could improve the water quality and the anti-disease ability of P.vannamei.