L-asparaginase produced by various microorganisms which it used for the treatment of leukemia and starchy food industry. Screening of L-asparaginase producing bacteria from soil samples from Damietta different locations was done. The primary screening by qualitative methods using rapid plate assay determined that out of 25 isolates, 20 strains were producing L-asparaginase. The greatest pink color zones were observed for four bacterial isolates(A6, A11, C11 and C19) after 48 hrs. These four isolates that preliminary screened were grown in liquid media for static and submerged fermentation and then used for secondary screening by Nesslerization method. The best four isolates producing L-asparaginase were identified based on morphological, cultural and biochemical tests, as A6, C11 and C19 were identified as Bacillus species and A11 was identified as Staphylococcus species.