Gene knockout, an important technology in molecular biology, has been broadly applied in industrial microbial metabolic engineering. From the basic mechanism of DNA recombination, we summarized and compared in this review different gene knockout strategies. Three most hot and important approaches, including the λ Red recombination system using the linear dsDNA as recombination substrate, the single or double crossover homologous recombination using the circular plasmid DNA as substrate, and the transposase mediated transposition recombination, were summarized in detail. Developing frontiers and application prospects of gene knockout were further discussed.