Karst area has specifically geological structure. The process of dissolution leads to notable surface features and typical karst landscape. Ecosystems in the karst environment are quite vulnerable to climate change and human activities. Identifying and quantifying the disturbance by a karst disturbance index could help resource managers formulate approaches and policies to reduce these anthropogenic impacts. As a world natural heritage site, the unique karst landscape in the Lijiang River Basin, Guilin City, China is of great values for tourism, ecology, and scientific research. However, in the coupling system of human activities and natural environment, multi-dimensional interferences threatened the Lijiang River Basin. In terms of the characteristics of the Lijiang River Basin and previous studies, a comprehensive evaluation system comprising geomorphic, hydrological, climatic, biological, and social factors, as well as 15 specific disturbance indexes was designed. The karst comprehensive disturbance index was calculated. The spatial correlation analysis was used to explore the explanatory power of geographic factors to comprehensive disturbance and the characteristics of the interaction force between factors and indicators. The results showed that: (1) the comprehensive disturbance index in the middle reaches of Lijiang River Basin was higher than that in the upper and lower reaches. The natural landform background and the zonal distribution of different types of karst landscape were main reasons for the difference. (2) The disturbance level was significant disturbance, and the value of comprehensive disturbance index was 0.336. The spatial distribution features were low in the east and north, and high in the west and south, with obviously spatial heterogeneity. (3) The geographical environment and socio-economic factors had strong explanatory power to the comprehensive disturbance, and some indicators such as quarries and mining, various disasters, production activities, and altitude had strongly spatial correlation. The karst disturbance index model was used to construct an evaluation system, and the feasibility of the model was verified, the degree and scope of the influence of multiple human and natural disturbances in the Lijiang River Basin were described, which could highlight the areas in urgent need of protection and the leading disturbance factors. The study evaluates the comprehensive disturbance of karst in the Lijiang River Basin and analyzes its spatial characteristics. The results can provide important reference value for the policy making of karst ecological environment protection and landscape resources sustainable development.