Proteases from Bacillus sp show a broad range of industrial applications due to remarkable properties they present including high stability and activity. The Bacillus sp. LBA 46 strain was selected as a high producer of protease and was identified as Bacillus licheniformis. Protease activity was measured using azocasein as substrate at pH 7.0 and 60°C. The study of protease production under submerged fermentation, using low cost agro-industrial substrates was performed utilizing response surface methodology (RSM). In the fermentation of the microorganism in Erlenmeyer flasks containing culture medium composed of 32 g/L of sugar cane molasses, 6 g/L of corn steep liquor, 2 g/L of yeast extract Prodex-Lac SD® at 30°C, 200 rpm for 96h it was obtained 868U/mL of protease. In a further study on the effect of milk powder concentration in protease production it was obtained the highest protease production (3000 U/mL) using medium composed of 40 g/L of sugar cane molasses, 6 g/L of corn steep liquor, 2 g/L of yeast extract Prodex-Lac SD® and 20 g/L of dried whey. The crude protease extract from Bacillus sp. LBA 46 obtained from the cultivation in optimized medium was applied in the removal of blood and tomato sauce with ground beef stains in cotton fabrics. The blood stains were completely removed when the fabrics were washed using the combination of the protease with laundry detergent. Furthermore, better results in the removal of tomato sauce with ground beef stains were observed using the combination of laundry detergent with proteases when compared to the use of detergent alone.