It is of great significance to explore the effect of ultrasonic treatment on the binding behavior of lutein (LUT) and bovine serum albumin (BSA) for the development of active molecular carrier. The interaction between LUT and BSA at different ultrasonic frequencies (0 Hz/40 kHz/53 kHz) was analyzed by fluorescence spectroscopy and UV-visible absorption spectroscopy. The binding sites were determined by binding site competition experiment and molecular docking technology. The results showed that LUT could quench the fluorescence of BSA at different ultrasonic frequencies， and the quenching rate was the highest (50.6%) at 53 kHz. Meanwhile， ultrasonic treatment could enhance the hydrophobicity of BSA structure， which was consistent with UV-vis absorption spectrum. Synchronous fluorescence showed that ultrasonic treatment made the BSA skeleton loose and the fluorescence intensity of tryptophan changed significantly at 53 kHz. Molecular docking showed that the binding site of LUT and BSA was located between subdomain ⅢA and ⅢB， which was verified by site competition experiments. In addition， thermodynamic studies and molecular docking showed that LUT spontaneously binds to BSA mainly through hydrogen bonding and hydrophobic interaction. In conclusion， ultrasonic treatment has a significant impact on the binding behavior of LUT and BSA at 53 kHz， which provides a new idea to regulate the interaction between protein and small molecules in food by changing the ultrasonic frequency.