Using 1-octadecyl ester-1, 2-benzenedicarboxylic acid (OEBA) as a collector, the flotation separation of fluorite, calcite and barite was investigated in this paper. The micro-flotation results showed that OEBA efficiently floated out fluorite and barite from calcite at pH ∼ 9.0, and barite from fluorite at pH ∼ 11.0. OEBA adsorbed on the surface of fluorite, barite and calcite through forming the Ca(Ⅱ)/Ba(Ⅱ)-carboxylate species. The adsorption capacity, in-situ AFM image, interaction force and adhesion force deduced that at pH ∼ 9.0, the hydrophobicity of the three minerals treated by OEBA was followed as fluorite > barite ≫ calcite, while it changed to barite > fluorite ≫ calcite at pH ∼ 11.0. These well explained the sequential flotation separation of barite, fluorite and calcite at pH ∼ 9.0 and 11.0. This work also provided the adhesion force criterion for designing and developing collectors, as well as the flotation separation conditions including the pulp pH, collector or depressant.