L-asparaginase has emerged as one of the most important clinically used enzymes as it exhibits chemotherapeutic potential in treatment of acute lymphoblastic leukemia and lymphosacroma. In the present study, soil microbial isolates were screened for potential producers of L-asparaginase using a phenol red indicator growth medium and the isolate producing the largest hydrolysis zone was selected, identified using 16SrRNA sequencing, and then named Stenotrophomonas sp. FZ. It was able to grow and produce L-asparaginase enzyme under submerged fermentation using shaken and static conditions (1.937 IU and 1.787 UI, respectively). Eleven culture conditions were examined for their significance on enzyme production using Plackett–Burman factorial design. Casein hydrolysate and magnesium sulphate were the most significant factors improving enzyme production. Maximal enzyme activity (21.23 IU) has been detected which is more than tree folds the activity recorded in basal medium. A verification experiment revealed more than 99% validity. The enzyme was partially purified by acetone precipitation with 73.32 % yield and a purification factor of 6.32 fold. Further purification includes gel filtration chromatography on Sephadex G-75 and C-25. The maximum enzyme activity was recorded at pH7 and 35°C. The effect of substrate concentration showed a progressive increase in the enzyme activity in a concentration dependent manner until it reaches a plateau where saturation was reached. The kinetics parameters, Km and Vmax, of Stenotrophomonas sp. L-asparaginase were 96.71 mg/ml and 3333.33 umol/ml/min, respectively.