<p>This review provides a comprehensive overview of the conformational changes between the open and closed states of loop structures within enzyme molecules and their significance in enzyme engineering. The article begins by introducing the developmental background of protein engineering and the structural characteristics of enzymes, with a particular focus on the pivotal role of loops in enzyme activity, substrate specificity, and environmental adaptability. Through instrumental analysis and computational approaches, the molecular mechanisms underlying loop opening and closing are thoroughly examined, and recent practices of loop engineering based on point mutations, directed evolution, and rational design are systematically summarized. The studies indicate that the dynamic changes of loops are central to influencing catalytic efficiency and specificity, and their modification can significantly optimize enzyme performance. Therefore, the in-depth research in this field not only provides novel perspectives for elucidating the dynamic processes of enzymatic catalysis but also paves innovative pathways for optimizing engineering strategies to develop high-performance artificial enzymes and green biomanufacturing technologies.</p>