The growing dependence on fossil fuels is one of the key factors that prove the urgency of the development and implementation of sustainable biofuels as alternative energy sources. Biofuels can also be made out of waste products of plants. Nevertheless, it needs vital enzymatic transformation, where natural enzymes do not always lend themselves to operational stability in order to implement and succeed in the industrial environment. Recent inventions on the immobilization method of enzymes have significantly enhanced the enzyme life and catalytic activity, which is offering a viable means of generating enzyme biofuels long-term. New opportunities have emerged in this field with nanotechnology, more so, the introduction of nanoparticles, nanotubes, and magnetic nanomaterials that come in handy in augmenting the enzyme surface attachment and stability in industries. The materials improve the enzymes in the sense that they help improve their performance and make it easy to carry out any reuse operation that leads to the optimization of producing biofuels that include biodiesel, bioethanol, and production of bio gases. This part gives the background knowledge on enzyme immobilization and explains various nanomaterials that are optimal in this method. The chapter describes technical specifications for testing system performance, which utilizes different laboratory equipment. The promising results are hindered by current hurdles, including operating expenses and the challenges of large-scale production, as well as protection concerns. The chapter explores emerging possibilities that combine intelligent nanocarriers with artificial intelligence systems for designing superior enzyme frameworks.