As an important biologically active substance, β-carotene has poor stability and low bioavailability, which greatly limits its application in food. Soybean protein isolate (SPI) was used as water phase, corn oil as oil phase to form a multilayer emulsion with chitosan and sodium alginate, which was cross-linked with Ca2+ to prepare β-carotene microspheres. The effects of mass fractions of chitosan and sodium alginate on the stability and β-carotene release characteristics of emulsion and gel microspheres were studied by analyzing the particle size, ζ-potential, emulsification stability, interfacial protein adsorption capacity, embedding rate of β-carotene, scanning electron microscope, Fourier transform infrared spectroscopy and in vitro release. The results showed that when the mass fractions of chitosan was 2.0%, the stability of the secondary emulsion was the best, and the embedding rate of β-carotene was up to (64.82±0.31)%, when the mass fractions of of sodium alginate reached 2.0%, the embedding efficiency of β-carotene in the three-layer emulsion was significantly increased, with the value of (86.75±2.00)%. The Fourier transform infrared spectroscopy (FTIR) indicated the electrostatic interaction between sodium alginate and chitosan. The dried gel microspheres were spherical and the structure of gel microspheres became more compact with the increase of sodium alginate concentration. In addition, in vitro release experiments showed that the gel microspheres had the function of continuous release. Emulsion gel microspheres can be used as a potential sustained-release system for β-carotene.