In this study, we explore the evolution and formation mechanism of innovation ecosystem niche fitness from three perspectives: theoretical analysis, model construction, and empirical testing. Based on the niche theory, a theoretical framework for the innovation ecosystem is constructed. Spatiotemporal analysis and qualitative comparison methods are employed to estimate the innovation niche fitness of nine provinces located in the Yellow River Basin, and their spatiotemporal characteristics and differences in terms of formation mechanism differences are then explored. The results show that: (1) temporally, from 2000 to 2017, the innovation niche fitness in the Yellow River Basin experienced minor fluctuations. However, since 2017, there has been a considerable increase. Spatially, the midstream and downstream regions, particularly in the provinces of Shandong, Henan, and Sichuan, have a higher innovation fitness. Conversely, the upstream regions of Qinghai, Ningxia, Gansu, and Inner Mongolia display a lower fitness because of the lack of advantages in innovation elements. (2) The innovation niche fitness is heavily influenced by several factors, including the number of industrial enterprises above designated size, R&D personnel, higher education institutions, scientific research institutions, expenditure for technical renovation, sales revenue of new products, total expenditure on R&D, and the total retail sales of consumer goods. (3) Four mechanisms explain high innovation niche fitness, each of which corresponds to different regions. The formation mechanisms of low innovation niche fitness can be categorized into two paths, which have an asymmetric relationship with the formation mechanisms of high innovation niche fitness. Therefore, provinces and regions should focus on these advantages to enhance the innovation niche fitness. Our research will serve as a theoretical reference to enhance the overall innovation capacity of the Yellow River Basin in the future.