L-Ascorbate oxidase (EC 1.10.3.3) is a vital enzyme found in various plant species, with cucurbits being a notable source, playing a crucial role in the oxidation of ascorbic acid to dehydroascorbic acid. The enzyme is of significant interest due to its essential role in producing ascorbic acid, a key product with wide-ranging applications in the agri-food and pharmaceutical industries. Given the importance of ascorbic acid in various domains, a deeper understanding of this enzyme is warranted. This study focuses on a thorough investigation of zucchini L-ascorbate oxidase, specifically from Cucurbita pepo medullosa. The methodology encompasses the extraction and purification of the enzyme, with a focus on maintaining structural integrity during these processes. The subsequent examination of kinetic parameters involved the determination of enzyme activity, substrate concentration, and reaction conditions. Our findings reveal a detailed characterization of L-ascorbate oxidase, with a particular emphasis on its structural composition. Spectroscopic properties of its copper atoms offer insights into the binding and reduction of substrates. The enzyme also contains carbohydrate residues, which contribute to its overall composition. These insights into the enzyme's structure provide a foundation for understanding its function. The research underscores the importance of L-ascorbate oxidase in the context of ascorbic acid metabolism and oxygen activation within biological systems. By exploring its structure, and kinetic properties, we contribute to a deeper comprehension of its functional significance. This understanding has implications for potential applications in diverse sectors, including agriculture, nutrition, and healthcare.