AbstractCoastal nitrogen enrichment significantly contributes to the decline of seagrass health and habitat, thereby diminishing its capacity to capture and sequester carbon (i.e., Blue Carbon). However, the consequences to blue carbon stocks due to sublethal changes in chemical recalcitrance of seagrass organic matter (OM) caused by nitrogen enrichment is unknown. In this study, we investigated the effects of nitrogen‐loading on the chemical composition of meadow‐forming Thalassia hemprichii. We found that the amino acid content in seagrass leaves and rhizomes increased with nitrogen loading, while the total labile OM and refractory OM (i.e., cellulose and lignin) content in seagrass leaves, sheaths, and roots generally decreased with nitrogen loading. Additionally, cellulose and lignin within sheath tissues showed a threshold response whereby refractory OM dropped when leaf nitrogen content (i.e., indicator of nitrogen loading) exceeded 2.2%. The rhizome labile OM and cellulose content also peaked at 2.2% leaf nitrogen content but subsequently reduced to a minimum at ~2.9% leaf nitrogen. The threshold of 2.2% leaf nitrogen content, equivalent to 43~72 μM dissolved inorganic nitrogen concentration in sediment pore water, may be utilized to forecast the contribution of seagrass refractory OM to sediments. It was estimated that high nitrogen loading could result in a loss of 309–645 kg ha−1 of refractory OM inputs in this study sites. The findings underscore that nitrogen enrichment has sublethal but significant negative impacts on carbon cycling via the reduction of refractory OM in seagrass biomass, consequently weakening the autochthonous contribution of seagrass to long‐term carbon sequestration.