Reducing dependence on non-renewable resources has driven the development of sustainable biotechnological processes, with industrial biotechnology playing a central role. This research focuses on the production of isoquercetin, a health-promoting compound, by optimizing and scaling up enzyme-based conversion of rutin using recombinant α-L-rhamnosidase. Technical development was complemented by techno-economic analysis, providing insights into the feasibility and sustainability of large-scale isoquercetin production.