Taking the Nandadish Watershed as the research object, the watershed runoff was divided into surface runoff(SR), subsurface 0~50 cm runoff (SSR50), and subsurface 50~100 cm runoff (SSR100) based on hydrological and geomorphological characteristics. Through testing hydrogen and oxygen isotopes and hydrochemical indicators in various water bodies (precipitation, soil water, groundwater, layered runoff) during typical rainfall events, combined with high resolution hydrometeorological monitoring data, the formation mechanisms of layered runoff under different underlying surface meteorological conditions were analyzed. Hydrological tracing techniques coupled with end member mixing models revealed the dynamic changes in contribution proportions of various water sources to layered runoff. Results show that layered runoff generation mechanisms and water sources differ significantly under various underlying surface meteorological conditions. Under wet multistage rainfall patterns, mixed runoff generation modes of infiltration excess and saturation excess predominate (runoff SR is higher than SSR100, which in turn is higher than SSR50). Under wet short duration intense rainfall patterns, surface infiltration excess runoff is dominant (runoff SR is higher than SSR100, which in turn is higher than SSR50). Under drought continuous rainfall patterns, there is a transition from infiltration excess to saturation excess runoff (runoff SSR100 is higher than SR, which in turn is higher than SSR50). The water sources of layered runoff generally show a pattern where, with increasing depth, the contribution proportion of event water decreases while that of pre event water increases. SSR50, serving as a transition layer for runoff stratification, has water sources significantly influenced by underlying surface conditions, exhibiting dynamic “dominant balanced” conversion characteristics.