The flotation separation of fine smithsonite and calcite has always been a challenging issue in the mineral processing industry. Both minerals are microfine particle distribution in nature, requiring ultra-fine grinding to maximize their liberation, which makes their flotation separation difficult. This study investigated the effects of PEO (Polyethylene oxide) and alum as combination agent on the flotation separation of fine smithsonite and calcite. The research results showed that using PEO and alum as combination agent can effectively separate fine smithsonite and calcite. PEO has a flocculation effects on both minerals, and Alum has better selective adsorption and flocculation effects on calcite, thereby effectively reducing the recovery of calcite. After treatment with PEO and alum, soluble starch tends to adsorb on the surface of calcite more than smithsonite, selectively depressing calcite in flotation separation, thus allowing for the effective flotation separation of fine smithsonite and calcite.