This review summarizes the latest progress in enzyme preparation, including enzyme design and modification technology, exploration of new enzyme sources, and application of enzyme preparation in food processing, detection, and preservation. The directed evolution technology improved the stability and catalytic efficiency of enzymes, while enzyme immobilization technology enhanced reusability and industrial applicability. Extremozymes and biomimetic enzymes exhibit excellent performance under harsh conditions. In food processing, enzyme preparation can improve food quality and flavor. In food detection, enzymes combined with immune detection and biosensors realize rapid detection of allergens, pollutants, and pesticide residues. In food preservation, enzymes enhance food quality by extending shelf life and inhibiting microbial growth. In the future, enzyme engineering will be combined with computer-aided design, artificial intelligence, and new material technology to promote intelligent enzyme design and multifunctional enzyme preparation development and help the technological upgrading and sustainable development of the food industry and green chemistry.