Denitrification is a major biological source or sink of N2O, an important greenhouse gas, which is a multi-step respiratory process that converts nitrate (NO3−) to gaseous forms of nitrogen (N2 or N2O). In aquatic ecosystems, the residence time and flow length within the streambed can modulate denitrification, which is highly affected by micro-topography of streambed. For the reason, various restoration projects targeting streams and rivers incorporate the installment of dune-shaped streambed, but its role in ecosystem functions and key controlling variables are still elusive. In this study, N2O fluxes according to streambed micro-topography were measured at four locations in two streams with dune-shaped streambeds in Korea. We found that micro-topographical variations influenced N2O fluxes, chemical properties, and denitrifier abundances, but that this effect was highly constrained by carbon availability. In sites with relatively higher DOC, N2O fluxes increased along the flow direction, and the highest flux of each site (19.1 ± 5.67 μg N2O-N m−2 h−1 and 16.2 ± 0.896 μg N2O-N m−2 h−1, respectively) was observed in the slope on the back side of the dune, followed by decreases afterward. In contrast, streams with low DOC concentration did not exhibit micro-topographical variations. This study suggests that the presence of small dunes in streams can substantially enhance N2O fluxes when carbon availability was appropriate. The variations of N2O flux highlight the importance of micro-topography and DOC concentration of sediment for the accurate estimation of uncertainty of N2O flux. This has further implication for stream restoration where installment or management of dune-shaped streambed is of importance in the removal of inorganic N in water.