Abstract: Gene knockout is a kind of gene modification technology by which a specific gene with known sequences but without functional information can be inactivated or deleted via experiment designed at molecular level so as to speculate the biological function of the gene. Currently, this technology has been employed in many fields： in establishment of animal disease models, the application of gene knockout develops rapidly and it was widely used in human, mice, zebrafish, chicken, bovine, sheep, porcine,and other species; in functional genomics, the functional information of genes is found by establishment of gene knockout animal models; in pharmacology, gene knockout mice can be used to reveal the mechanism underlining the pathogenesis of diseases and to discover novel therapeutic targets; in immunology, the immune system diseases can be investigated at genetic level by gene knockout; in prevention and treatment of animal diseases, gene knockout is also wildly applied; in transgenic animal research, a lot of regulatory elements have been investigated for the external regulation of transgenic animals; gene knockout technology is also employed in research regarding replication, metabolism and pathogenesis of pathogenic microorganisms as well as growth,development and breeding of animals.