Abstract: Metabolic engineering is the combination of applied molecular biology and reaction engineering.It has become the platform technology for strain improvement.In the post-genomic era, genomics, transcriptomics, proteomics and metabolomics provide great opportunity for the development of metabolic engineering.In addition to new pathway construction, DNA shuffling and genome shuffling are applied to rapid optimization of known or unknown pathway, which greatly promotes the development and applications of metabolic engineering. DNA shuffling and genome shuffling methods can increase our understanding of metabolic networks and their molecular regulation mechanisms，integrating and complementing rational metabolic design strategies. This review illustrates the applications of DNA shuffling and genome shuffling in metabolic engineering and prospects the perspective and methodology in metabolic engineering by genome shuffling.