At the scale of geomorphological units (riffles, pools, and gravel bars), the contribution of the hyporheic zone to the functioning of streams and rivers depends on the hydrological exchanges between surface water and groundwater. These exchanges are largely controlled by sediment structure and texture, which are difficult to assess with classical methods (shovelling and freeze coring). We aimed to evaluate the ability of a non-destructive method, ground-penetrating radar (GPR), to detect sediment structures associated with subsurface biologically active zones in a gravel-bed river. After GPR data acquisition and processing, a three-dimensional reconstruction of a gravel bar permitted the identification of two sediment facies: a cobble/gravel lithofacies denoted as ‘coarse’ and a sand/gravel lithofacies denoted as ‘fine’. We installed piezometers along two longitudinal profiles (each corresponding to a lithofacies identified by GPR) and monitored hydraulic head and temperature for 20 days. Water and sediments were sampled along the two profiles to measure water physicochemistry, sediment characteristics, bacterial abundance and activity, and interstitial invertebrate assemblages. These measurements confirmed that the two profiles were characterized by distinct hydrological flow rates and associated biological activities. Rapid water transport in the coarse profile fuelled the hyporheic zone with organic matter, whereas water and organic matter supplies were lower in the fine profile. Consequently, the higher supply of organic matter in the coarse profile was associated with higher microbial activities and invertebrate density and diversity. Therefore, GPR could be an efficient tool to detect the sediment features playing a key role in hyporheic zone functioning.