Ecotourism is vital for coordinating regional ecological protection with socio-economic development. The Gansu section of the Yellow River Basin is a typical ecologically fragile area in China, and it holds a distinctive position in ecological protection and high-quality development. This study explores spatial differentiation in ecotourist perceptions and their distinct effects on ecotourist satisfaction, revisitation, and recommendation. It uses four cities (Gannan, Linxia, Lanzhou, and Baiyin) in the Gansu section of the Yellow River (mainstream) as examples, employing a questionnaire survey to collect ecotourists’ perception data and applying a random forest model and one-way ANOVA for analysis. It was found that: (1) rich ecotourism potential exists in the Gansu section of the Yellow River Basin as an ecologically fragile area; (2) there is spatial differentiation in ecotourist perceptions, and among the four regions, Baiyin stands out for its nature and atmosphere perception, and Lanzhou excels in accessibility and service perception; (3) spatial disparities exist in the influencing factors of ecotourist satisfaction, revisitation, and recommendation. Ecotourists in districts with unique natural resources, such as Gannan and Baiyin, prioritize nature perception, whereas districts with abundant natural resources and an established foundation for ecotourism development, such as Linxia and Lanzhou, emphasize service and atmosphere perception. This study constructs a new research framework to explore spatial variations in ecotourists’ perceptions, assisting ecotourism destinations to meet the needs of ecotourists from the supply side, and presents distinctive strategies and recommendations for the development of ecotourism in similar ecologically fragile areas.