Protease producing bacterium was isolated from the fish gut of Oreochromis mossambicus and identified as Lysinibacillus fusiformis F3G5 by 16S rRNA gene sequencing. The nutritional factors such as carbon sources, nitrogen sources, ionic sources and also environmental parameters such as temperature, pH, sodium chloride were optimized to enhance the yield of protease. Among the carbon sources, fructose (439.27±15.14 U/ml) enhanced enzyme production. Ammonium sulphate (439.27±15.14 U/ml) was found to be the best nitrogen source and Zn 2+ ion (662.70±23.32 U/ml) significantly influenced on enzyme production. Protease production was found to be high at pH 7.0, 40 °C at 0.5% NaCl and the partially purified enzyme was highly active under these environmental conditions. The partially purified enzyme showed more than 60% activity in the presence of a few detergents. The demonstrated detergent stability of protease from L. fusiformis F3G5 infers that the enzyme has good compatibility with ingredients present in detergent formulation.