Recently, growing concern has been paid to the toxicity of additives in food. The present study investigated the interaction of two commonly used food colorants, quinoline yellow (QY) and sunset yellow (SY), with catalase and trypsin under physiological conditions by fluorescence, isothermal titration calorimetry (ITC), ultraviolet-vis absorption, synchronous fluorescence techniques as well as molecular docking. Based on the fluorescence spectra and ITC data, both QY and SY could significantly quench the intrinsic fluorescence of catalase or trypsin spontaneously to form a moderate complex driven by different forces. Additionally, the thermodynamics results demonstrated QY bind more tightly to both catalase and trypsin than SY, suggesting QY poses more of a threat to two enzymes than SY. Furthermore, the binding of two colorants could not only lead to the conformational and microenvironmental alterations of both catalase and trypsin, but also inhibit the activity of two enzymes. This study provides an important reference for understanding the biological transportation of synthetic food colorants in vivo, and enhancing their risk assessment on food safety.