Soil borne fungi produce a broad variety of extracellular enzymes and protease is one of them. Alkaline protease extracted from fungal isolates (named as BY-1 and BY-2) was purified by precipitation with different concentration of ammonium sulfate. A purified enzyme extract obtained from isolates (BY-1 and BY-2) revealed increased protease activity as compared to the initial crude extract. Enzyme fraction obtained from isolate BY-1 after 70% saturation of (NH 4) 2 SO 4 demonstrated higher protease activity (32.02 U mL-1) as compared to its initial crude extract (31.84 U mL-1) and it was named as enzyme fraction P1. Enzyme fraction obtained from isolate BY-2 after 80% saturation of (NH 4) 2 SO 4 revealed increased protease activity (16.77 U mL-1), when compared to the activity (9.15 U mL-1) in its initial crude lysate and it was named as enzyme fraction P2. Partially purified enzyme was used for characterization study. Protease activity from both enzyme fractions (P1 and P2) was highest when the pH of the reaction mixture was adjusted to 8 and 9, indicates alkaline nature of protease. Optimum temperature for protease activity by both the enzyme fractions (P1 and P2) was found to be 50 ºC, indicates thermostability of enzyme. Ca 2+ ion increased protease activity by 7.15% and 23.05% from fraction P1 and P2, respectively. Protease activity from fraction P1 and P2 was stimulated by Ca 2+ and Mn 2+, respectively. n-butanol increased protease activity from enzyme fractions P1 and P2 by 5.70% and 35.71%, respectively, while methanol and acetone revealed inhibitory effect on protease activity.