In this study, β-carotene loaded oil-in-water emulsions were stabilized by complex interfaces composed of propylene glycol alginate (PGA), rhamnolipids (Rha), and zein colloidal particles (ZCPs). The influence of mixed biopolymer–surfactant, biopolymer–particle, surfactant–particle and biopolymer–surfactant–particle interfaces on the performance of the emulsions was investigated. The stability, microstructure, rheological properties, and in vitro gastrointestinal digestion of the emulsions were controlled by regulating the adding sequence and mass ratio of the multiple stabilizers. The droplet size of the emulsion was in the range of 14–77 μm. After encapsulation into the emulsions stabilized by the complex interfaces, the photothermal stability of β-carotene were increased by 41.53% and 21.52%, respectively. The co-existence of particles, biopolymers, and surfactants could induce competitive displacement, multilayer deposition and an interparticle network at the interface. Compared with a single PGA- or Rha-stabilized emulsion, the complex interface-stabilized emulsion reduced the release of FFA by 28.06% and 26.16%, respectively. The interfacial composition of the emulsion and the delayed lipid digestion further affected the bioaccessibility of β-carotene in the gastrointestinal tract (GIT). The mixed biopolymer–particle–surfactant interface-stabilized emulsion could be incorporated in foods, pharmaceuticals and cosmetics for excellent stability, targeted nutrient delivery and controlled lipolysis.