Although rational method and global technique have been successfully applied in strain improvement respectively, the demand for engineering complex phenotypes required combinatorial approach. The technology of genome shuffling has been presented as a novel whole genome engineering approach for the rapid improvement of cellular phenotypes. This approach using recursive protoplast fusion with multi-parental strains offers the advantage of recombination throughout the entire genome without the necessity for genome sequence data or network information. Genome shuffling has been demonstrated as an effective method, which is not only for producing improved strain but also for providing information on complex phenotype. In this review we attempt to present the advantage of genome shuffling, introduce the procedure of this technology, summarize the applications of this approach for phenotype improvement and then give perspective on the development of this method in the future.