An excellent alternative for chemical antimicrobial agents to prevent disease in the shrimp aquaculture is the application of effective probiotics. The present study was evaluated the effect of Bacillus vallismortis IS03 as a native probiotic, isolated from digestive tract of Litopenaeus vannamei on pathogenic Vibrio harveyi under in vitro and in vivo circumstances. Co-cultivation of V. harveyi and B. vallismortis showed significantly (P<0.05) decreased the growth of V. harveyi in the treatment groups compared to the control. Cell-free extracts of B. vallismortis IS03 exhibited more appropriate antibacterial effects on replication of V. harveyi. The highest and lowest inhibitory effects were respectively shown in10⁸ and 10⁶ CFU ml^-1 of B. vallismortis IS03 cell-free extracts. The probiotic potential of B. vallismortis IS03 was assessed through the groups of control and the experiments 10⁶, 10⁷ and 10⁸ CFU ml^-1 salt water once every 3 days from zoea1 process to end point of the study. The probiotic potential of B. vallismortis IS03 was assessed through the groups of control and the experiments 10⁶, 10⁷ and 10⁸ CFU ml^-1 salt water once every 3 days from zoea1 process to end point of the study. Shrimp survival was determined after 10 days of challenge with V. harveyi  at 10 ⁵ CFU ml^-1 (for the first 5 days) and 10 ⁷ CFU ml^-1  (for the second 5 days).The cumulative mortality in the treatment with 10⁸ CFU ml^-1 of  B. vallismortis IS03 reached 11.88% compared to 40.63% in the control group. At the end of the trial, total bacterial counts on TSA, total vibrio on TCBS were significantly (P<0.05) lower in the 10⁸ CFU ml^-1 treatment group. Bacillus counts on MYP agar in the treatment groups were significantly (P<0.05) higher than the control, also total bacterial counts was lower in the treatment groups, while, no vibrio were grown in the muscle tissues of shrimp treated with probiotics. It is concluded that 10⁸ CFU ml^-1 of probiotic, B. vallismortis IS03 has antibacterial efficiency against pathogenic V. harveyi at in vitro and in vivo conditions.