A clinical bacterial isolate from a patient urine sample in Kasr El-Aini was identified by biochemical and molecular means to be E. coli . This isolate was optimized for production of L-asparaginase (L-asparagine amidohydrolase), a relatively widespread enzyme found in bacteria, eukaryote and mammals but not man. This enzyme catalyzes the deamidation of L-asparagine to L-aspartic acid and ammonia. The production of L-asparaginase was achieved through optimization of fermentation parameters and it showed 6.05 IU of enzyme activity. The produced L-asparaginase was then purified by means of chromatography techniques and tested against three different cell lines for its anticancerous activity, human colon cancer CACO-2, Human breast Cancer MCF-7 and Human cancer prostate PC-3. The expression for the regulatory genes BAX, P53 and BCL2, was analyzed by RT-PCR and it was clear that L-Asparaginase enzyme shows anticancer activity against (MCF-7) and (PC-3), where it was non-effective to the cell line (CACO-2). It was also noticed that BAX and P53 genes were upregulated under the effect of Asparaginase enzyme and that BcL2 gene was down-regulated in Human Breast and prostate Cancer cell line while Human colon cell line was not.