Production of protease by Trichoderma estonicum isolated from mangrove sediment was statistically optimized for culture conditions using a two-step approach. Statistical modeling method of Plackett-Burman was used for identification of the important factors that are responsible for protease production while the central composite design was used for further optimization of the important factors. For the identification of the important factors for protease production, six factors such as fructose (g/L), paraffin liquid (%), NaCl (g/L), CaCl2 (g/L), temperature (°C) and pH were tested. Among these, three factors - fructose (g/L), NaCl (g/L), and temperature (°C) - were found to be important for protease production in addition to that one important factor was incubation period also added for the further optimization. After statistical optimization of production medium (fructose 0.5 g/L, NaCl 3.22 g/L, temperature 41°C), protease production was 41.54 IU/mL which was 66.57% higher than the normal production medium in 107 hours of incubation. The crude protease enzyme extract was tested for the blood stain removal. The highest removal activity of 59.7% was observed in enzyme extract, which was greater than that of the commercial detergent with 55.1% removal activity. Therefore Trichoderma derived enzyme is a promising detergent in removal of blood strain.