In this study, anti-leukemic enzyme L-asparaginase (E.C.3.5.1.1) from Escherichia coli ATCC 11303 was modified by the microencapsulation technique onto calcium alginate beads. Using response surface methodology (RSM), a three-level full factorial design, the values of concentration of sodium alginate, concentration of calcium chloride, and enzyme loading were investigated to obtain the highest residual L-asparaginase (L-ASNase) activity % (immobilized enzyme activity/free enzyme activity). The effects of the studied factors on immobilization were evaluated The predicted values by the model were close to the experimental values, indicating suitability of the model. The results presented that an increase in sodium alginate concentration increased the percent of residual activity of L-ASNase at any given calcium chloride concentration and the moderate amount of enzyme loading increased the percent residual activity. The optimal immobilization conditions were as follows: sodium alginate 1.98% (w/v), calcium chloride concentration 3.70% (w/v), and enzyme load 46.91% (v/v). The highest residual L-ASNase activity % obtained was 34.49%.