Soil denitrification enzyme activity was measured by a hierarchical sampling approach within the riparian zone of the Liu Xi River tributary of Guangzhou city, and explored and identified multi-scale factors that influenced soil denitrification. The results showed that soil denitrification potential—represented by soil denitrification enzyme activity (DEA)—showed more heterogeneity on a profile-scale than on a cross-sectional scale and landscape-scale in riparian zones; multi-scale factors controlled the degree of spatial variation in soil denitrification. The profile-scale factors, including soil TOC (total organic carbon), TN (total nitrogen) and NO3--N (nitrate nitrogen), were the major direct regulators of the spatial distribution of DEA. The cross-sectional scale factors, including landscape position and vegetation density, and the landscape-scale factors, including TI, elevation and land-use types, indirectly regulated the distribution of DEA. At the profile scale, the highest DEA occurred on the upper soil, but dramatically declined from the soil surface to the deeper layers. At the cross-sectional scale, the highest DEA was observed at the landscape position, with good landscape connectivity and high-density vegetation within the middle part of the riparian zone. At the watershed scale, DEA showed an increasing trend from upstream to downstream except for the sites in urbanized areas.