Abstract: Restoring the natural habitat of Gymnocypris przewalskii is an important strategy to protect its resources. Among the essential elements of this habitat, riverbed substrate stands out, underscoring the necessity to discern the substrate preferences of G. przewalskii for habitat creation. Through a comprehensive habitat survey, this study identified five typical substrate types. Subsequently, a simulation pond featuring these substrate was constructed in the Gangbei Canal of Shaliu River, a Qinghai Lake tributary. Employing quantitative preference index methods, we evaluated the substrate preferences and intensity of both male and female G. przewalskii individuals. Additionally, we analyzed the internal and external factors influencing substrate selection in G. przewalskii. The results showed that (1) Females G. przewalskii exhibited the longest visit durations on gravel substrate (2540s), while males favored small and medium pebble (1960s); (2) Females displayed a preference solely for sandy and gravel substrate (PI=6595.8), whereas males preferred all three substrate types, particularly small and medium pebbles (PR=10.3); (3) Male G. przewalskii individuals tended to follow females’ substrate preference in group selection, with gravel substrate attracting the longest visit durations (2051s). This study aims to provide a theoretical basis for river substrate modification and the creation of “natural” habitats conducive to G. przewalskii.