Nowadays, one of the most important family of drugs, are enzymes. The major sources of enzymes in biotechnology are plant tissues and organs of animals. L-Asparaginase enzyme is an enzyme that exists in chicken liver, and because of its medicinal use in cancer treatment is a valuable enzyme. In general, separation of dissolved and undissolved materials from diluted solutions is a difficult task. For substances such as proteins and enzymes this problem becomes more serious. That is due to the fact that these materials are sensitive to changes in operating conditions such as temperature and shear rate. Unfortunately, there is not an ideal method for the purification of enzymes. The enzymes purification usually involves a multistep process that is associated with the loss of large amounts of enzyme activity. Therefore, search for development of new methods for purification of enzymes with sufficient selectivity and efficient recovery and enrichment that can preserve enzyme activity with acceptable costs and short process times is continued. On the other hand, nano and micro-bubbles have attracted much attention in recent decades. This is because they have been used in various fields of science and technology. They have been used applications in water purification, separation processes, friction reduction, and minerals processing, to name but a few. The objective of this research was to extract L-Asparaginase from chicken liver using nano and micro-bubbles, and examine the effects of the pertinent parameters on the efficiency of extraction. Hence, in this study, nano and micro-bubble separation method is examined for extraction and enrichment of L-asparaginase from the chicken liver. In this method, the separation is based on adsorption of enzyme onto the gas-liquid interface. To produce nano and micro-bubbles, a solution containing AOT surfactant was agitated by ultrasonic agitator and with homogenizer with turbine blades at speeds of 8000 rpm and 2500 rpm for 5 minutes, respectively. Then the enzyme solution was added and mixed with bubbles. The concentration of the enriched enzyme solution was measured by UV apparatus. The enrichment and the evaluation of the lack of denaturation of L-asparaginase was performed by electrophoresis. Dynamic light scattering (DLS) was used to determine the size distribution of bubbles. The average of the bubble size distribution when produced at 8000 rpm, in presence of KCl, and a concentration of 6 mM of AOT, and a pH of 6 was about 12 nm. Various surfactants were examined. The results showed that AOT as an anionic surfactant was the most favorable surface active agent. This surface active agent could produce nano-bubbles. The effects of the pertinent parameter such as concentration of AOT, presence of salt, ionic power, and pH of the solution were studied. Potassium chloride and sodium sulfate were used. Presence of potassium chloride demonstrated better results. The results showed that at an agitation speed of 8000 rpm, a pH of 6, and a concentration of 6 mM AOT, and when the average size distribution of bubbles were 12 nm, the highest enrichment was achieved. Finally, results of electrophoresis showed that the separation by nanobubbles have not altered the molecular structure of L-asparaginase. Keywords: AOT, L-Asparaginase enzyme, nano and micro-bubbles, protein purification, chicken liver, purification, aphron