Collectors are critical in the flotation and separation of fine-grained minerals. In this study, a new combined collector, namely phosphoric acid (SPA)/dodecylbenzene sulfonate isopropanolamine (DBIA) was prepared via self-assembly. First, the flotation of cassiterite was investigated using SPA and DBIA separately, and the micro-collection mechanism of these single-material collectors on the surface of cassiterite was clarified by density functional theory calculations. Then, combined SPA/DBIA collectors were prepared, and the effect of the SPA/DBIA molar mass ratio on cassiterite flotation performance was studied. Surface tension measurements and molecular dynamics (MD) simulations were used to investigate the gas–liquid interfacial self-assembly of the SPA/DBIA collector. Subsequently, the flotation separation behavior of SPA/DBIA on the solid–liquid–gas interface was experimentally evaluated, and the adsorption mechanism of this combined collector at the three-phase interface was comprehensively investigated by performing MD simulations. The results show that SPA/DBIA combined collectors can obtain 80.88 % cassiterite flotation recovery at 1.2 × 10−4 mol/L. The addition of SPA led to the continuous adsorption of DBIA on the surface of cassiterite, forming a more stable and dense adsorption film. The mass density of the adsorption film was 319.27 ng/cm2, owing to the synergistic co-adsorption. Finally, the surface of the cassiterite was modified to enhance its hydrophobicity. This work demonstrates the application of collector self-assembly to improve the recovery of fine-grained cassiterite via flotation.