Aquaculture industry especially shrimp farming in Iran and other parts of the world has been facing various disease that among various agents, pathogenic bacteria has emerged as important because it causes large economic losses to the shrimp farming industry. This study investigated the antagonistic properties of copper nanoparticles (34 mg/L), probiotic Bacillus subtilis (108 CFU/mL), and combination of these two against with pathogenic bacteria, luminescent Vibrio harveyi in vitro condition in order to investigate possible use of probiotic B. subtilis and copper nanoparticles in the diet for growth and immunity enhancement in Litopenaeus vannamei. This study reports that in Well diffusion test and Disc diffusion test against pathogenic bacteria luminescent V. harveyi maximum inhibition showed by combination of suspension of probiotic B. subtilis and copper nanoparticles the amount of (W = 22.54±1.72 and D = 16.37±2.9) whereas minimum inhibition was copper nanoparticles the amount of (W & D = 0). Results indicated the possible synchronic use of probiotic B. subtilis and copper nanoparticles in shrimp diet.