Caveolin-1 (CAV-1) plays a key role in developing cardiovascular diseases such as atherosclerosis. The interaction between quercetin and CAV-1 was studied by multispectral, homology modeling, molecular docking simulation and bio-layer interferometry (BLI) in the simulated physiological environment and different temperatures. The fluorescence quenching data showed that the Kq value (the quenching rate constant) were all much larger than 2.0×1010 L·mol-1·s-1, and the fluorescence quenching constant (KSV) decreased with the increase of temperature, which proves that the quenching process of the interaction between quercetin and CAV-1 is static quenching. Furthermore, the thermodynamic parameters, enthalpy change ΔH＜0, entropy change ΔS＜0 and ΔG＜0 indicated that the bonding process is spontaneous and enthalpy driven, indicating that the main types of interaction are van der Waals force and hydrogen bonding. Through the synchronous fluorescence and three-dimensional fluorescence spectrums analysis of the interaction between quercetin and CAV-1, it was found that the fluorescence intensity of CAV-1 was progressively decreased upon the addition of quercetin, indicating that quercetin interacted with CAV-1. Further analysis showed that quercetin caused the redshift of the maximum emission wavelength of the aromatic amino acid residues in CAV-1, enhanced the polarity of the microenvironment around the CAV-1, enhanced its hydrophilicity, indicating that the addition of quercetin changed the protein conformation of CAV-1. The UV-Vis absorption spectrum showed that a ground-state complex was formed between CAV-1 and quercetin, which further confirmed the static quenching mechanism between CAV-1 and quercetin. The X-ray crystal structure template of CAV-1 was constructed using homology modeling. The molecular docking simulation results showed that the binding force of quercetin and CAV-1 was -7.372 kcal·mol-1. The docking results showed that quercetin could bind to the active pocket composed of amino acids such as Glu20, ASP70, VAL16 and ARG19. There were the van der Waals forces between quercetin and residues GLN21, VAL16 and ARG19 of CAV-1, and hydrogen bonds between quercetin and GLU20 and ASP70. Various forces affected the micro-environmental changes of CAV-1 and led to its fluorescence quenching, which is a key factor involved in the formation of the complex. Finally, the binding of quercetin and CAV-1 was quantitatively studied by the BLI technique. The results showed that quercetin had a good binding activity with CAV-1 with an equilibrium constant (KD) value of 2.50×10-5 mol·L-1. The response signal value increased with quercetin concentration, evidencing the specific binding between CAV-1 and quercetin. This research was helpful in understanding the mechanism of interaction between quercetin and CAV-1 and provide references for research on the therapeutic targets of quercetin in atherosclerosis.