The use of some species of Bacillus as a probiotic has been widely reported. In general, using probiotics in the exact isolation location will improve the performance of these probiotics. This study aims to characterize the Bacillus subtilis (BR610) isolate, successfully isolated from the intestines of Rabbitfish previously cultivated in the floating net of Awerange bay, Barru, South Sulawesi. The research includes morphological characterization of BR610 isolates, activity tests of amylase, cellulose, and proteinase enzyme. BR610 isolate inhibition test against several types of pathogenic bacterial isolates (Vibrio harveyi, V. parahaemolyticus, V. alginolitycus, and Streptococcus) as well as BR610 isolate resistance test against various kinds of antibiotics. The results showed that the Bacillus subtilis (BR610) has a spherical morphology, wavy edges, flat elevation, and is cream-white in color. BR610 can produce the enzyme amylase 20.36±17.8mm, proteinase 14.2±6.7mm, and Cellulose 20.9±2.7mm. These isolates can also inhibit the growth of V. harveyi (13.0±0.3mm), V. alginolyticus (15.1±2.8mm), V. parahaemolyticus (14.7±2.7mm), and Streptococcus (17.6±1.6mm). Bacillus subtilis BR610 is still worthy of being used as an environmentally friendly probiotic because it is still sensitive to several types of antibiotics tested.