Taking 288 cities in China as research samples,the business environment index, soft-environment of doing business index and hard-environment of doing business index of 288 cities in China from 2014 to 2020 are constructed, and the data of 288 cities in China from 2013 to 2019 are matched. The effect and mechanism of high-speed rail opening to improve the urban business environment are discussed by using the double difference method. The results show that the opening of high-speed railway has effectively improved the urban doingbusiness business environment, soft-environment of doing business and hard-environment of doing business. The product of the nearest port distance and the number of high-speed railways opened at the provincial level is selected as the instrumental variable for robustness test, and the conclusion is still valid. The moderating mechanism shows that the Advancement of urban industrial structure enhances the positive relationship between the opening of high-speed rail and the business environment, while the rationalization of urban industrial structure has no significant impact on the relationship between the opening of high-speed rail and the business environment. The study explores the motivation of business environment optimization, and also deepens the understanding of regional business environment optimization, mechanism and regional differences under the background of large-scale transportation infrastructure empowerment.