Purified asparaginase that’s highly purified extracted from frozen plant seeds of Pisam sativum var J of. were used to determine the specificity of enzyme against different substrates. Enzyme specificity was 783.1, 28.1, 11.4, and 13.7 U/mg of L-aspargine, L-aspartic acid, L-glutamine, and L-glutamic acid respectively. The cytotoxicity effect of purified asparaginase of frozen seeds of Pizum sativumvar J of. against L20B tumor cell line measured at wave length of 450, 492 and 620nm respectively, it showed that’s purified asparaginase has maximum inhibitory effect on tumor cell line reached to 33, 33, and 23% growth at 450, 492 and 620nm respectively. Cell survival and remaining activity of L20B tumor cell line after treatment with purified asparaginase reduced to 68, 65 and 77% respectively after incubation with purified asparaginase at concentration of 150 µg/ml, while the cell line remaining activity percentage reduced to 66.6, 67.7 and 76µg/ml respectively.