Bioretention basins are a prominent type of vegetated stormwater infrastructure that provides various ecosystem services, such as carbon (C) sequestration. Despite the key role of organic matter in the performance of bioretention basins, there is little understanding of their C accumulation properties. Using detailed field studies, we investigated the spatial, temporal and vertical variation of C capture in the soil of 25 subtropical bioretention basins in Australia. A thirteen-year soil chronosequence was used to estimate C sequestration rate. It was observed that the bioretention basins displayed a spatially uniform depositional pattern of C in their ponding area. The mean areal C density of soil in the upper 20 cm was 3.8 ± 0.3 kg C m−2, from which 32% was associated with the top 5 cm of soil. There was a strong influence of age on C density only throughout the first top 20 cm of the soil profile with a C sequestration rate of 0.31 kg C m−2 yr−1. Carbon quickly accumulates in the top 5 cm layer while in the lower depths it accumulated at a more gradual rate. The results show that bioretention systems could be designed for the enhancement of their C sequestration potential, and amendments in their design, such as addition of a carbon source layer, are important for better managing carbon availability in the basins.