Abstract: The hydrothermal carbonization of microalgae has attracted much attention in recent years because it is a relatively mild process that can produce high-quality carbon materials such as N-doped carbon dots (CDs) without additional additives. However, the multicomponent coexistence of microalgae has led to complex hydrothermal reaction paths, and the interaction between components is unclear, which cannot effectively improve the yield and performance of CDs, becoming a core challenge for the controlled synthesis of microalgae-based CDs. In this study, glucose and glycine were selected as model compounds for carbohydrates and proteins to systematically investigate the effects of HTC parameters (temperature: 190−250 ℃, time: 2−8 h) on the synthesis of CDs. The results showed that the mass yields of glucose-based and glycine-based CDs prepared under the hydrothermal conditions in this study did not exceed 4.05 mg/g, whereas the interaction reaction between glucose and glycine could significantly enhance the mass yield of CDs, which could reach up to 29.85 mg/g. The fluorescence properties of glucose-based CDs were poor, with fluorescence quantum yields of less than 0.50% under all conditions, whereas the in situ N-doping of glycine-based CDs can reach 1.67% of fluorescence quantum yields. During the interaction reaction between glucose and glycine, not only retained hydrophilic groups such as —OH and —COOH on the surface of CDs, but also successfully doped with N in the carbon core, which made the glucose/glycine-based CDs with both high mass yield and fluorescence quantum yield. The effects of reaction temperature and time on the hydrothermal reaction of carbohydrate-protein model compounds were revealed, and it was found that the interaction between glucose and glycine could significantly improve the mass yield of CDs, and the N-containing compounds such as proteins could promote the N-doping of CDs to enhance the fluorescence performance. This study provides theoretical support for the characterization of the interaction reaction between the main components of microalgae and the design of high-performance N-doped CDs.