L-ASPARAGINASE (L-ASNase) has been widely used as a therapeutic agent in the treatment of various lymphoblastic leukemia diseases. This study aimed to isolate and purify local bacterial isolates that are capable of producing L-ASNase, so 150 bacterial isolates from the NileRiver where isolated, purified and their ability to produce L-ASNase was assessed. Among these isolates, 32 bacterial isolates showed their ability to produce L-ASNase, so they were selected for further studies. The most active bacterial isolate in the production of L-ASNase was selected, identified as Escherichia coli and named MG27. Enzyme localization was determined in cultures grown under aerobic and anaerobic conditions. E. coli MG27 was found to produce more L-ASNase in anaerobic conditions compared with that produced under aerobic conditions with 128 folds.  Finally, in an attempt to determine the optimum conditions for periplasmic L-ASNase production, the influence of several cultural factors was investigated.