The impact of lactoferrin (LF)–chlorogenic acid (CA) and (−)-Epigallocatechin-3-gallate (EGCG) conjugates on the physicochemical properties of β-carotene emulsions was investigated. Formation of lactoferrin–polyphenol conjugates, which was confirmed by SDS–PAGE, caused changes in the structure and nature of lactoferrin. Based on layer-by-layer electrostatic deposition, β-carotene bilayer emulsions were prepared by lactoferrin and lactoferrin–polyphenol conjugates at pH 7.0. The physicochemical properties of primary and secondary emulsions were evaluated and the results suggested that LF–polyphenol conjugates-stabilized primary and secondary emulsions exhibited better emulsifying properties and improved physical stability of β-carotene bilayer emulsions under freeze–thaw, ionic strength and thermal treatments. In addition, the lactoferrin–polyphenol conjugates could effectively enhance chemical stability of β-carotene in oil-in-water emulsions against heat treatment and ultraviolet light exposure, and the least degradation of β-carotene occurred in LF–EGCG conjugate-stabilized primary emulsion. The interfacial engineering technology utilized in this study may lead to the formation of emulsions with improved physicochemical and functional performance.