Functional dairy products are rich in bioactive ingredients like polyphenols which are beneficial to human body， but at the same time， in order to improve the appearance of food， the addition of synthetic azo pigment amaranth (Ama) increases the health risk of food to some extent. In this study， fluorescence spectroscopy combined with molecular docking was used to study the binding competitive effect of five polyphenols and Ama with β-lactoglobulin (β-Lg). The results showed that curcumin (Cur)， resveratrol (Res)， quercetin (Que)， caffeic acid phenylethyl ester (CAPE)， chlorogenic acid (CGA) and Ama could form complexes with β-Lg. The binding constants of several ligands with β-Lg were in the order of 105~107 L/mol， which indicated that the binding ability was very strong. Among them， Ama and CGA bind β-Lg by hydrogen bond and van der Waals force， while Cur， Res， Que and CAPE bind β-Lg by hydrophobic interaction. Under acidic conditions， Ama and five polyphenols could bind to β-Lg at different sites， and no obvious competitive binding effect was observed. Under neutral condition， Ama and Cur competed for binding site of β-Lg. When polyphenols were first bound to β-Lg， Res affected the binding of Ama to β-Lg， that was， the order of ligand addition affected the competitive effect. At the same time， both Cur and Ama bind to the site at the entrance of β-Lg hydrophobic cavity， and both of them bind with Asn90， and the competitive effect was stronger than Res. In this paper， the competition between small molecules and milk protein was studied， which provided a theoretical basis for the practical application of controlling the absorption of some food additives by means of processing.