Several researches have focused on the enzymatic pretreatment of lignocellulose biomass to produce fermentable sugars that can lead to ethanol production thus facilitating pathways for sustainable biofuel production. Enzymes are fundamental to the pretreatment process, however, are required in larger quantities during pretreatment process thus influencing biofuel production cost. Immobilization of enzymes to a suitable support/matrix could enhance its stability, and reusability thus containing cost. This chapter focuses on developing an advanced technology for immobilizing enzymes to nanomaterials; variety of nanomaterials used for immobilization, nature of enzyme/protein nanomaterial interactions, methods of enzyme immobilization, and factors affecting mode of interaction for achieving hydrolysis of microcrystalline cellulose and natural cellulosic substrate. The binding of enzyme (94%) to a nanomaterial was established by spectroscopy techniques. The kinetics study, conducted at optimum pH (pH 4) and temperature (50 °C for free and 60 °C immobilized enzyme), exhibited improvement in immobilized enzyme properties. The immobilized enzyme retained up to 50% of its enzyme activity in five consecutive cycles. This chapter advocates the use of nano-immobilized enzymes in biomass hydrolysis for biofuel production.