Abstract Extensive reservoir construction has fragmented more than 70% of the world's rivers, significantly impacting river connectivity and carbon cycling. However, the response of riverine dissolved organic matter (DOM) to reservoir influence and its potential downstream effects remains unclear. In this study, we employed multiple analytical techniques, including Fourier transform ion cyclotron resonance mass spectrometry, radiocarbon dating, and environmental factor analysis, to investigate the dynamic changes in DOM and its controlling factors under different hydrological management regimes in the LongTan Reservoir, the largest reservoir in the Pearl River, which is the second largest river in China by water discharge. Our results indicate that the molecular diversity of riverine DOM is reduced in the reservoir. Oxygen‐rich and heteroatomic compounds, such as those containing nitrogen, sulfur, and phosphorus, are preferentially removed through enhanced photo‐ and biodegradation processes in the reservoir, particularly during the storage period. This leads to DOM that is enriched with oxygen‐poor compounds and shows a biodegraded Δ 14 C value downstream. This study highlights that the composition of riverine DOM is significantly altered by the reservoir, but these effects could potentially be mitigated by optimizing the outlet location.