The efficient removal of dolomite from phosphate ores is vital for the subsequent production of phosphate fertilizer. This work investigated the interaction mechanism of poly(sodium styrene sulfonate) (PSS) on apatite and dolomite surface and its implication on flotation through Micro-flotation, adsorption capacity, Zeta potential, SEM-EDS and density functional theory simulation. Flotation results proved that PSS was able to depress dolomite efficiently in broad pH range (8–12). Contact angle, Zeta potential, adsorption capacity and SEM-EDS measurement confirmed that PSS was more prone to adsorb on dolomite rather than that on apatite, which hindered sodium oleate (NaOL) to adsorb on dolomite but almost didn’t influence NaOL to interact with apatite surface. DFT calculations confirmed that the 2p orbital of oxygen atoms within sulfonate groups hybridized with 4s orbital of calcium and 3s orbital of magnesium atoms on dolomite surface to form OCa, OCa and OMg covalent bond, while PSS weakly physisorbed on apatite via electrical interaction, due to the significant difference in steric hindrance and active atom density. PSS exhibited good selectivity and environmental compatibility as dolomite depressant in the beneficiation of phosphate ores for sustainable production of phosphate fertilizer.