In this paper, a feasible eco-hydrological regionalization methodology was presented for determining the target of ecological protection and ecological water requirement in a basin. The eco-hydrological regionalization of a watershed was defined based on a review of related researches; according to ecological importance, hydrological characteristics, ecological background and effects of human activities of the Yellow River basin, the indicator system of regionalization was constructed. Firstly the values of the rule hierarchy were calculated by the indicator hierarchy with county as unit, which was easy for management; then class regionalization of every county was decided by the corresponding levels of the rule hierarchy; the Yellow River basin was divided into 20 first-class zones and 51 second-class zones in the end. The protection preferential levels of second-class zones were determined by RIAM model with ecological importance as indicators, 2 significant preferential reserves and 11 moderate preferential reserves need to be protected especially. In the watershed, the preferential reserves mainly distributed in the headwater area, between the east of Helan Mountain and the west of Ordos, the juncture of Shanxi Province, Shaanxi Province and Henan Province, and the delta.