In order to reveal particulate organic carbon (POC) source and mode of transport in the largest river basin on Earth, we sampled the main sediment-laden tributaries of the Amazon system (Solimões, Madeira and Amazon) during two sampling campaigns, following vertical depth-profiles. This sampling technique takes advantage of hydrodynamic sorting to access the full range of solid erosion products transported by the river. Using the Al/Si ratio of the river sediments as a proxy for grain size, we find a general increase in POC content with Al/Si, as sediments become finer. However, the sample set shows marked variability in the POC content for a given Al/Si ratio, with the Madeira River having lower POC content across the measured range in Al/Si. The POC content is not strongly related to the specific surface area (SSA) of the suspended load, and bed sediments have a much lower POC/SSA ratio. These data suggest that SSA exerts a significant, yet partial, control on POC transport in Amazon River suspended sediment. We suggest that the role of clay mineralogy, discrete POC particles and rock-derived POC warrant further attention in order to fully understand POC transport in large rivers. To examine the source of POC in the Amazon Basin in more detail, we use radiocarbon (14C) content and the stable isotope composition (δ13C) of POC. 14C activity demonstrates that rock-derived POC is a significant component of river bed sediments and contributes to POC across the river depth-profiles of suspended sediments. We estimate that the flux of rock-derived POC may reach ~ 10% of the total POC export by the Amazon River. After correcting for rock-derived POC input, we find that POC from the terrestrial biosphere (biospheric POC) is mostly sourced from C3-plants. Higher biospheric POC δ13C values in the Madeira River (by ~ 0.5–1‰) are best explained by a small (<5%) contribution of C4-grasses from Bolivian savannas. Finally, we use 14C to estimate the mean age of biospheric POC exported from the Amazon Basin. The data show that biospheric POC is younger in the Solimões River (1120 years) than in the Madeira River (2850 years). These ages are much younger than the corresponding estimates of sediment residence time in the basin, suggesting that lowland areas and/or young POC from above-ground biomass may contribute disproportionately to the biomarker signals in terrigenous sediments of the Amazon delta.