The enzyme L-asparaginase has been a clinically acceptable anti-tumor agent for the effective treatment of acute lymphoblastic leukemia. In the present study, the purified L-asparaginase from Streptomyces gulbargensis was immobilized in three different matrices such as calcium alginate, gelatin and alginate-gelatin fibers. The activity yields obtained with calcium alginate, gelatin and alginate-gelatin fibers was 39%, 44% and 69% respectively. Hence, entrapment in alginate-gelatin fibers was found to be comparatively superior to the other two matrices. Characterization of the alginate-gelatin entrapped L-asparaginase revealed that the immobilized enzyme showed a shift in optimum pH value from pH 9.0 (optimum pH value of free enzyme) to 9.5. However, there was no change in the optimum temperature for the immobilized enzyme compared to the free enzyme, being 40^oC for both forms of enzymes. Further, there was a significant increase in the pH and temperature stability of the immobilized L-asparaginase compared to the free enzyme.