A fungal strain identified as Penicillium sp. was evaluated for its L-asparaginase enzyme production. The L-asparaginase enzyme was purified to homogeneity from Penicillium sp. that was grown on submerged fermentation. Different purification steps including salt precipitation, followed by separation on sephadex G-100-120 gel filtration and DEAE were applied to obtain pure enzyme preparation. The purified enzyme showed 13.97 IU/mg specific activity and 36.204% yield. The polyacrylmide gel electrophoresis of the pure enzyme exhibited one protein of 66 kDa. The enzyme showed maximum activity at 7.0 pH and 37°C and K m value 4.00 × 10-3 M.