Abstract: Wild-type strains hardly meet the current industrial demands due to low stability. The performance of microbial strains (high-yielding, high-quality strains) can be improved by using physical mutagenesis techniques. At the same time, high throughput screening methods and techniques can be used to quickly obtain ideal strains from the mutation library. However, traditional manual screening and shaker culture are high-cost, time-consuming, and laborious, and high-throughput screening technology can solve this problem. In this review, the applications of traditional and novel mutagenesis used for the improvement of probiotics are summarized, the principles of traditional and novel physical mutagenesis techniques are discussed, the differences between the two kinds of mutagenesis techniques are compared and their applications in the improvement of probiotics are described. At the same time, the characteristics and relevant applications of various high-throughput screening technologies (microtitration plate screening, fluorescence activated cell sorting, biosensors screening, droplet microfluidic platform screening and model animal platform screening) are summarized. This review provides important reference for reducing screening cost, improving screening efficiency and obtaining high yield ideal target strains.