In recent years, the potential impact of food-derived carbon dots (CDs) produced during the thermal processing of foods on human health has attracted the attention of many people. In this study, CDs were extracted and purified from beef roasted at 280 ℃ for 30 min and evaluated for its interaction with digestive proteases (pepsin and trypsin) by in vitro simulated digestion, fluorescence spectroscopy, synchronous fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy and thermodynamic analysis. The results of simulated digestion in vitro showed that the CDs could interact with digestive proteases. Fluorescence spectroscopic analysis showed that the CDs statistically quenched the inherent fluorescence of digestive proteases. The synchronous fluorescence results demonstrated that tyrosine was involved in the interaction. The thermodynamic results showed that the binding mode of the CDs and digestive proteases was electrostatic or hydrophobic interaction, and the combination affected the secondary structure of pepsin and trypsin. After interaction with the CDs at 1.0 × 10^-5 mol/L, the activities of pepsin and trypsin decreased to (61.11 ± 7.36)% and (51.28 ± 3.62)% of their original value, respectively.