Abstract: The investigation on river water isotope characteristics of small watersheds and the sampling scheme are of great significance for a better understanding of the precipitation-runoff relationship, and for the establishment of an isotope hydrological station network. This study conducted high-frequency sampling in the main control section of the Huashan watershed, which is the representative watershed of the Jianghuai transition zone, and its adjacent nested sub-watershed outlet sections for a whole hydrological year. The annual variation in hydrogen and oxygen isotopes in river water was analyzed to provide a basis for the development of river water sampling schemes for small watersheds. The results showed that: ① The hydrogen and oxygen isotopes in river water for the two sections of the Huashan watershed varied consistently, but the isotopes in the main control section were higher than those in the tributary outlet section. ② The hydrogen and oxygen isotopes in river water showed four stages of variation from the flood season, namely the fluctuation and decline period in summer, the rising period in autumn, the stable period in winter, and the rising period in spring. ③ River water isotopes showed three types of response to heavy rainfall, namely rapid decline during mid-heavy rainfall, gradual decline during mid-heavy rainfall, and gradual decline during late heavy rainfall. ④ The recommended routine sampling scheme for river water is to collect samples every 2 days in summer and every 15 days during other seasons. The recommended sampling scheme for river water during the rainy season is to sample the section water every 4 hours, from the beginning of rainfall until the end of rainfall for heavy rainfall in summer, autumn, and spring. These research findings may provide a reference for the sampling of river water isotopes in China's mid and small watershed station networks.