<p>Immobilization of enzymes is a key strategy for industrial biocatalysis; however, preserving catalytic activity during repeated use remains a major challenge. In this study, we report an ionic liquid (IL)-assisted preparation strategy for lipase–copper hybrid nanoflowers (lipase-Cu hNFs) that enables enhanced enzymatic activity and reusability. Lipase-Cu hNFs were synthesized by introducing imidazolium-based ILs during the biomineralization process, thereby modulating the local microenvironment of the enzyme without altering the conventional nanoflower architecture. Systematic evaluation of IL concentration, alkyl chain length, and anion type revealed that an optimal amount of IL significantly improved the initial catalytic activity and effectively retained enzymatic performance over repeated reaction cycles. Among the tested ILs, [Bmim][TfO] provided the most balanced enhancement, affording higher activity retention compared to other anions, which exhibited either limited stabilization or rapid activity loss upon reuse. Notably, the [Bmim][TfO]-mediated hNFs exhibited higher catalytic activity at 30 and 60&#xa0;°C and maintained more than 50% of their initial activity after five consecutive cycles. Overall, this work demonstrates that IL-assisted preparation offers a simple and effective route to improving the activity and reusability of lipase-based hNFs, highlighting its potential as a practical strategy for enhancing the operational performance of immobilized enzymes.</p>