In order to examine the impacts of water-sediment regulation on regional carbon cycling, we collected water, particulate and sediment samples from the middle-lower Yellow River in late June and early July, 2015 and analyzed their specific amino acids (AA), DOC, POC, and bacteria abundance. Summarized by 14 specific AA, the total hydrolysable AA (THAA), particulate AA (PAA), and sediment AA (SAA) varied in ranges of 2.29–9.05 µmol L –1, 5.22–22.96 µmol L –1, and 81.7–137.19 µg g –1 dry weight. After the regulation, dissolved free AA (DFAA) decreased by 29% while DCAA increased by 72%. These variations suggested that DFAA were further degraded, while DCAA molecules were further activated. Meanwhile, PAA increased almost 4 times as many as those before regulation, and SAA increased as well. After regulation, the amounts of bioactive amino acids (Asp, Glu and Gly) increased in THAA but decreased in PAA, with little changes in SAA. The ratios of Asp/Gly in different phases increased after regulation, indicating the AA contributions were promoted by calcareous organisms rather than by siliceous organisms. Multiple correlation analysis showed that PAA was primary representatives of AA and organic carbon, followed by DCAA and POC. Moreover, bacterial reproduction played a key role in shaping the AA compositions and properties, followed by the redox condition and acid-base balance. The results of this study provided a clear evidence for the effects of water-sediment regulation on regional biogeochemistry of organic carbon in the middle-lower Yellow River.