Abstract: In the present study, we report the cloning of L-asparaginase II (asnII) gene from E. coli W3110 into pGEX-2T DNA vector. The L-asparaginase II enzyme (E.C.3.5.1.1) was overexpressed in E. coli BL21(DE3) and purified to homogeneity 238.4 fold by utilizing chromatography technique on DEAE-Sepharose fast flow, Glutathione S sepharose 4B columns and thrombin. SDS-PAGE of the purified enzyme revealed that has Mr of 40 kDa. In addition, we found that the enzyme can be efficiently immobilized in calcium alginate gelatin composites. The free enzyme has an optimum pH at 7.5 but this optimum pH is shifted to 8.5 for the immobilized enzyme. The optimum temperature, for free and immobilized enzyme were 37 and 50°C, respectively. The immobilized enzyme retained most of its activity at 60°C with high stability compared with the native enzyme when incubated at 60°C for 30 min.