Aspergillus sp. is considered to be a good source of protease. Medium optimization is essential for each isolate in order to maximise the protease production. In the present study, different media were evaluated for maximising protease biosynthesis under submerged fermentation (SmF) using A. nidulans LCJ249 and A. flavus LCJ253. Among the six media tested, Medium 6 was found to be the best medium for maximum protease production in both A. nidulans LCJ249 (1178.0 U/mL) and A. flavus LCJ253 (950.6 U/mL). Effect of essential nutrient parameters like carbon, nitrogen and casein and other culture conditions such as incubation time, inoculum size, pH and shaking and static conditions were evaluated by the conventional one factor at a time approach. In the case of A. nidulans LCJ249, a medium containing 20 g/L glucose, 15 g/L malt extract, 5 g/L casein, initial medium pH of 7 and inoculum size of 30 mg/L favoured maximum protease production. Similarly, in A. flavus LCJ253, a medium containing 20 g/L starch, 20 g/L peptone, 15 g/L casein, initial medium pH of 7 and inoculum size of 10 mg/L favoured maximum protease production. In both the cultures, protease production was higher under shaking condition than in static conditions. The study showed that the optimised medium with optimal conditions enhanced protease production when compared to the original medium.