In the usual process of chromatography, the first step is the trapping stage, which attaches to the absorbent surface while the impurities do not bind. Subsequently, proteins that are weakly bound are removed prior to changing the conditions in order to wash the desired protein. Different Types of liquid chromatography methods are used to purify the protein. The only gel chromatographic process or molecular sieve chromatography is somewhat different because the isolation is based on differences in the size of proteins and the size of the Stationary phase cavities, not on the basis of surface absorption. The enzyme L-asparaginase accelerates the conversion of L-asparagine to L-aspartic acid and Ammonia. The enzyme L-asparaginase is one of the most important therapies in technology and biomedical field. The enzyme is used to treat acute lymphoblastic leukemia, chronic lymphoblastic leukemia, myeloid leukemia, melanosarcoma, and patients with Hodgkin apos;s lymphoma and non-Hodgkin apos;s lymphoma. Additionally, L-Asparaginase is produced by removing acrylamide, which is caused by the high temperature in starchy foods. Reduces the risk of cancer. The purpose of this study was to investigate the effective parameters in the purification of L-asparaginase enzyme from its rich solution from chicken liver through sedimentation and gel chromatography. In the sedimentation stage, two polyethylene glycol 4000 in the range of 10%, 20%, 40% and 60% and ammonium sulfate in the range of 40%, 60% and 80%, the highest sedimentation rates, for 20% weight polyethylene glycol 4000 and 60% ammonium sulfate with a purity were obtained. To design the experiment, modeling the data and optimizing the operating conditions of the chromatography column, Taguchi method was used in the Minitab 17 software. Gel chromatography column for two types of G-75 superfine gel and G-100 gel in the range of 10-30 height, pH: 6-8, flow rate of 15-30 ml/hr in three conditions including: without sediment, in the presence of optimal precipitator of ammonium sulfate and optimal precipitator of polyethylene glycol were investigated and the optimum conditions for superfine column G-75 with a height of 30 cm, pH=8, flow rate of 20 ml/hr and in the presence of a polyethylene glycol precipitator were achieved. Electrophoresis was used to determine the presence of the enzyme and purification function.