Abstract—In the present study production of L-asparaginase enzyme by Penicillium citrinum Thom. was determined in different culture and nutritional conditions. The fungal culture preferred fructose and ammonium nitrate for better enzyme production. Finally, the L- asparaginase extracted from cell biomass was purified through gel filtration and ion exchanged chromatography and characterized for its purity, molecular wt., thermal tolerance, pH requirement. Gel filtration and ion exchange chromatography produced pure enzyme which was later confirmed through PAGE exhibited 29.0 kDa. The partially pure enzyme preferred 6.0-7.2 pH, enzyme activity lowered down in higher pH. It was thermally stable under the treatment of 45-50 C. All experiments had been done under batch culture. However, the medium composition acquired finally can be useful for the continuous fermentation for large scale production of L - asparaginase.