Gene disruption by homologous recombination is a powerful tool for investigating gene function in yeast. Since 1980’s, it has been developed a lot. PCR-mediated gene disruption technique makes the manipulation easier and it can be used to precisely delete genes in yeast. Multi-gene disruption technique can delete several genes successively in the same yeast strain. After the completion of the yeast Saccharomyces cerevisiae genome sequencing, the gene disruption technique for systematic analysis meets the need of the functional genomics in yeast. It also enlighten the study on human functional genomics.