Abstract: Sanger sequencing method, first-generation sequencing method, has provided a powerful tool for human to decipher genetic codes. After more than 30 years′ continual improvement, second-generation sequencing method with some main characteristics of high throughput, rapid sequence and low cost has developed, and it brings revolutionary changes for life science field. The differences between second-generation sequencing and Sanger sequencing method were contrasted in this paper, and the application of second-generation sequencing in life science research was described.