Urban stream features can be used to promote nutrient retention; however, their interactions with different hydrological regimes impact nutrient cycling, decrease their retention capacity, and inhibit stream ecosystem functioning. This study analysed the temporal and spatial dynamics of the uptake of three nutrients (nitrate, ammonium, and phosphorus) in an urban drainage stream during high flows. In particular, we studied variations in net uptake along the right margin (with native vegetation and a roots mat) comparatively to the left margin (a non-rooted grassy bank). Applying the spiralling approach within each subreach on either side, we determined nutrient subreach (sr) retention metrics: uptake rate coefficients Ksr net, mass transfer rates vfsr net, and areal uptake rates Usr net. Our results showed nitrate (NO3) and ammonium (NH4) net uptakes on the right side were higher and more frequent along subreaches where the root mat was more abundant Usr net [μg m−2 s−1] = 22.80 ± 1.13 for NO3 and 10.50 ± 0.81 for NH4), whereas on the left side both nutrients showed patchy and inconsistent net uptake patterns despite the homogeneous grass distribution. Net uptake for filtered reactive phosphorus (FRP) was not observed on either side at any flow rate. The impact of hydrological factors such as discharge, travel time, water depth, and concentration, on uptake metrics was studied. Despite increases in travel time as the flow decreased, there was a reduction in net uptake rates, vfsr net and Usr net, on either side. This was attributed to a reduction in water level with declining flows, which decreased hydrologic connectivity with the stream banks, combined with a decrease in water velocity and a reduction in nutrient concentrations. We concluded the rooted bank acted as an effective retention area by systematically promoting net uptake resulting in a twofold increased dissolved inorganic nitrogen (DIN) retention relative to the non-rooted side where net uptake was spatially localized and highly dynamic. Overall, this work emphasized the importance of strategically sampling close to biologically active surfaces to more accurately determine areas where gross uptake surpasses release process.