L-asparaginase is a therapeutic enzyme that converts L-asparagine into ammonia and L-aspartate. L-asparaginase is used to treat acute lymphoblastic leukaemia. In food manufacturing industries, it is used to inhibit the acrylamide formation. The current investigation has been performed to isolate L-asparaginase producing fungi from different medicinal plants and soil samples, through serial dilution. A total number of 15 fungal isolates were obtained from soil samples and 6 endophytic fungi isolated from medicinal plants. By performing screening of L-asparaginase 67% of positive isolates were obtained from endophytes and soil samples. Optimization of L-asparaginase production was performed for parameters such as pH, temperature, carbon and nitrogen source, and it was found that pH 6, 30˚C, 2 g of glucose, and 1 g of L-arginine is suitable for maximum enzyme production. By performing Sodium dodecyl sulphate polyacrylamide gel electrophoresis the molecular weight of an enzyme was determined to be approximately 11.2 kDa.