To elucidate the interaction between carotenoids and human serum albumin (HSA) will provide theoretical basis for the development and utilization of active ingredients and the design and delivery of new drugs. In order to explore the interaction between carotenoids and HSA， fluorescence quenching effects of lutein (LUT)， β-carotene (β-CA)， lycopene(LYC) on HSA were analyzed by fluorescence spectroscopy， synchronous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy. Finally， the binding site was determined by site Marker experiment and molecular docking. The results showed that LUT， β-CA， LYC could quench the endogenous fluorescence of HSA， and β-CA had the highest fluorescence quenching rate for HSA. The binding constant of LUT， β-CA， LYC to HSA was in the order of 104-106 L/mol， and the binding ability of β-CA to HSA was the strongest. Thermodynamic parameters showed that LUT， β-CA， LYC and HSA bind mainly through hydrophobic interaction. Synchronization and three-dimensional fluorescence showed that the interaction between LUT， β-CA， LYC and HSA could change the spatial conformation of HSA. Marker assay and molecular docking technique showed that LUT， β-CA， LYC was bound near Sudlow's site I of HSA. The experimental results showed that all three carotenoids could bind to HSA， which would be of great significance for the development， utilization and targeted delivery of carotenoid activity.