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Cancer is a leading cause of death worldwide. In recently cancer caused 8.2 million deaths, and cancers of the lungs, liver, colon, stomach, and breast are main types. L-asparaginase belongs to the group of hydrolytic enzymes that is responsible for hydrolysis of L-asparagine into L-aspartic acid and ammonia. It had been used as an effective therapeutic agent against anticancer activity. In current study partially purified L-asparaginase enzyme from Aspergillus oryzae was tested in vitro anticancer activity by using MTT assay. To determine anticancer activity, different concentration of partially purified L-asparaginase was tested on Hep G2 and Hela cancer cell lines by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The enzyme showed a significant antiproliferative activity and a dose dependent effect was observed. Minimum viability of 2.60% was shown by enzyme at concentration 0.1μg/ml and maximum viability (66%) was observed at 2μg/ml. From the result it is conclude that this L-asparaginase can be used for the development of new preparations for the therapy of tumors.