This study aims to determine the potential of Bacillus subtilis probiotic in reducing levels of ammonia, Vibrio sp, and increased production performance in seaworm cultivation (Nereis sp.) under laboratory scale. Observation of the performance of seaworms (Nereis sp.) was carried out every 10 days which included weight gain (gr), length (cm), total biomass (gr), total bacteria (CFU/mL), total Vibrio sp. (CFU/mL), and total Bacillus subtilis (CFU/mL). Water quality measurements include temperature, dissolved oxygen (DO), pH, ammonia, and total organic matter (TOM). Application of probiotics Bacillus subtilis has the potential to reduce ammonia concentration, increase growth, and reduce the abundance of Vibrio sp under laboratory-scale seaworm cultivation. P3 treatment (0.01 mL with a probiotic density of 10 6 CFU/mL) gave the best results by being able to reduce the ammonia concentration by 47.5%. In summary, the probiotic application using the bacteria Bacillus subtilis with different densities able to provide good results in supporting production performance, maintaining the abundance of Vibrio sp., and reducing ammonia concentration in seaworm cultivation. This is the first study to report the performance of seaworm production using probiotic agent, research is still needed to determine the digestive enzyme activity of seaworms given probiotics.