Proteases possess some characteristics of biotechnological interest due to which these have become the most important industrial enzymes. The present study reported production of thermostable protease and its characterization using Bacillus species which is a thermotolerant bacterium, whereas Bacillus subtilis, is an organism widely used for isolating protease enzyme. Gelatin is used as substrate in nutrient agar medium for screening purpose, this showed the maximum zone of activity (22 mm) after overnight incubation period when the indicator was added. Under submerged fermentation condition high level of protease production occurred at 45 °C after 36 hours at pH-10, with continuous agitation (180 rpm). The presence of galactose and peptone in the medium enhances enzyme production by 0.5% when compared to the other carbon and nitrogen sources. Thus such additions can augment protease production and their applications in various industries.