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There is an increasing interest in using l -asparaginase in medical fields and food processing industries. Enzyme immobilization is an attractive field to improve l -asparaginase activity and stability. Graphene oxide (GO) is a promising candidate for enzyme immobilization due to its large specific surface area. In this study, GO was first functionalized with l -aspartic acid (GO-Asp), and then l -asparaginase was immobilized on the GO-Asp either physically or through chemical conjugation. A significant enzyme loading was achieved through covalent immobilization (100% immobilization efficiency). Stability of free and the immobilized l -asparaginase was examined at various temperatures (20–60 °C) and pH (5–9). The covalently immobilized l -asparaginase showed higher enzyme activity than free enzyme at pH 8 with the maximum recovered activity of 100%, 90.5% and 40.6% after 24 h of incubation at 20 °C, 40 °C and 60 °C, respectively. In addition, the covalently immobilized l -asparaginase on GO-Asp showed 42% recovered activity after eight continuous reaction cycles at 60 °C. The kinetic parameters of the immobilized and free enzyme were also calculated, indicating no significant changes in the enzyme affinity through covalent conjugation. The results clearly reflect the suitability of GO-Asp as a nanosheet support for l -asparaginase loading as well as its usage in future industrial applications.