The effect of flooding on soil enzyme activities and soil organic carbon (SOC) dynamics remains a widely debated topic. Here, we investigated spatial variations in C-hydrolyzing enzyme activities, soil C contents in different fractions [i.e. labile and recalcitrant carbon (LC and RC)] from 6 sites with four different elevations at two soil depths (0–10 cm and 10–30 cm) in riparian zones of the Three Gorges Reservoir, China. At region scales, the SOC, RC contents, and RC/SOC (RIC) generally showed decreasing tendency from the upstream to the downstream. The C-hydrolyzing enzyme activities were higher in the midstream compared to other sites, which did not correspond well with the changing trend of SOC content, but matched with the spatial variation in LC content. At ecosystem scales, the RC and RIC declined with decreased elevations, but the LC showed opposite trend. Whereas, the four C-hydrolyzing enzyme activities and the specific enzyme activities were corresponded well with the changing trend of LC content. Soil C contents and enzyme activities were generally higher in top soil than deep soil across sites and elevation zones. These results reveal that the LC is the tightest factor in regulating C-hydrolyzing enzyme activities, whereas the soil C quality (i.e. RIC) and flooding collectively drive C-hydrolyzing enzyme activities possibly by affecting decomposition rates of SOC in the riparian zones.