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*Corresponding author: Fanda Furlan Gon?alves Dias, Department of Food Science, School of Food Engineering, University of Campinas, P.O. Box 6121,13083-862 Campinas, SP, Brazil. Tel:+551935212175 Fax:+551935212153 E-mail:fandafgd@gmail.com,Objective: To explore the anti-proliferative activity of purified L-asparaginase from Aspergillus oryzae CCT 3940 (A. oryzae). Methods: L-asparaginase was produced by submerged fermentation and purified to electrophoresis homogeneity by ionic exchanged chromatography in a fast protein liquid chromatographic system. The purified enzyme was characterized and used for the anti-proliferative assay against nine tumor cell lines and one non-tumor cell line. Results: The free glutaminase L-asparaginase was purified 28.6 fold. L-asparaginase showed high stability under physiological condition, remaining stable in the pH range 7.0–8.0 after 1 h incubation at temperature range 30–45 °C. The Km and Vmax values of purified L-asparaginase were estimated as 0.66 mmol/L and 313 IU/mL, respectively. The purified enzyme could inhibit the growth of a broad range of human tumor cell lines at the concentrations studied. Also, the enzyme from A. oryzae CCT 3940 could inhibit tumor growth of leukemia cell line (K562) with a total growth inhibition value of (3.2 ± 2.5) IU/mL and did not inhibit the non-carcinogenic human cell line growth at the concentrations studied. Conclusions: The sensitivity of the cells lines to purified L-asparaginase from A. oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial L-asparaginase from Escherichia coli. The L-asparaginase from A. oryzae CCT 3940 has a high potential for pharmaceu-tical exploitation in the treatment of leukemia.