In this study is aimed the improvement of Bacillus subtilis E6-5 for high protease production by EMS treatment as chemical mutagen. The 82 mutants were screened on skim milk agar plates and, the mutant with maximum protease activity was named KE20. The mutant was quantitatively tested, and showed 9.2 times more protease activity than the parental strain. The effects of nutritional and physical factors on the protease production of mutant KE20 were investigated. The best carbon and nitrogen sources were corn starch and skim milk. No effect was observed when metal ions were used alone. In the physical parameters, the best results were obtained at 37°C, pH 7.0, 150 rpm, 3% as inoculum size and 18 has inoculum age. A new medium was made by optimizing the incubation conditions and enzyme yield enhanced 79% compared to basal medium. The mutant KE20 strain may have a prominent potential for protease production on an industrial scale.