In the present study, Streptomyces gulbargensis and its mutant form, S.gulbargensis mu24, immobilized on polyurethane foam were investigated for the production of L-asparaginase using groundnut cake extract as medium. The medium with an initial pH of 8.5 was inoculated with free and immobilized cells separately and then subjected to fermentation by incubation at 40oC and shaking at 200rev/min. In the immobilized cell system, enzyme production was enhanced by approximately 30% compared to the conventional FreeCell fermentation. The immobilized cells were subjected to repeated batch fermentation processes to determine their reusability. These cells retained their ability to produce L-asparaginase over seven cycles and the activities remained between 16.2-41.3 IU/ml and 39-60 IU/ml for S.gulbargensis and S.gulbargensis mu24, respectively. The maximum enzyme titer was obtained during the third batch by both strains. However, the mutant strain was more potent for L-asparaginase production than the prototype. Therefore, the polyurethane foam immobilized cells of S.gulbargensis mu24 can be proposed as an effective biocatalyst, which can be repeatedly used for maximum production of L-asparaginase.