Proteases are one of the enzymes required that have a significant impact on biotechnology. Bacillus subtilus MS1, a bacterial strain obtained from sewage sediment that could produce protease enzyme on skim milk agar. The isolate was identified as Bacillus subtilus MS1 with accession number KY621344 based on biochemical tests, microbiological, and 16S rRNA gene sequences. Various cultural conditions were optimized to become the maximum yield of enzyme production of 9.322 U/ml. Based on the optimization results, a significant scale culture was created utilizing glucose as a carbon source and peptone as a nitrogen source at 50°C for 48 h. Purification of the protease was achieved using 60% ammonium sulphate and Sephadex G-100 with a molecular weight of 30 kDa. The protease's activity maximized yields 24.437 U/mg at pH 10 and 50°C, and it remained stable at pH 6.0-11.0 and 30–80°C. In the presence of phenylmethyl sulphonyl fluoride (PMSF) and silver ion (Ag+1) the protease activity was suppressed. The purified protease enzyme occurred a greater stain removal.