Based on the data from 35 major cities in China in 2020, this paper applies the Simpson’s diversity index, the entropy value method, and the coupling coordination degree model to comprehensively measure the coupling coordination level of mixed land use and urban vitality in major cities in China and further analyze their spatial distribution characteristics. In addition, this paper analyzes the factors affecting the spatial variation of the coupling coordination level with the help of the geographic probe model. The study finds that: (1) The overall level of coupling coordination between mixed land use and urban vitality is high in 35 major cities in China. There is no disorder between mixed land use and urban vitality. (2) In terms of the spatial distribution of the coupling coordination between mixed land use and urban vitality in 35 cities in China, five cities, namely Beijing, Shenzhen, Shanghai, Guangzhou, and Chengdu, have the highest level of coupling coordination between mixed land use and urban vitality, reaching “good coordination” with a discrete spatial distribution. Central cities such as Hangzhou and Nanjing have the second highest level of coupling coordination and are at the “intermediate coordinate” with a “strip-like distribution” in space. Twenty cities in the north and south have the lowest coupling coordination levels and are in the “primary coordination.” Among these twenty cities, seven cities in the south have a higher level of coupling coordination than thirteen cities in the north, with a spatial distribution of a “C” shape. The northern cities have the lowest level of coupling coordination, with a “W”-shaped distribution in space. (3) Population size plays an essential role in guiding the level of coupling coordination between mixed land use and urban vitality in major cities in China, followed by government regulation and economic level. At the same time, transportation conditions and industrial structure have the weakest influence on the level of coupling coordination between mixed land use and urban vitality in major cities in China.