Although the effect of surface roughness on the floatability of a single mineral has been investigated extensively, a systematic study for the effect of surface roughness on the flotation separation of different minerals, e.g., flotation recovery, has never been dwelled. To fulfill this gap, in this study, the effect of surface roughness on the flotation separation of hematite from quartz was investigated with a series of experiments and detections. Micro-flotation experiments demonstrated that hematite particles with higher roughness resulted in higher recoveries for both hematite and quartz, but the improvement of hematite recovery was more significant, which promoted the flotation separation of hematite from quartz with a higher separation index (SI). Adhesion force measurements revealed that after interacting with 125 mg/L sodium oleate at pH 9, increasing nano-scaled roughness improved the hydrophobicity of hematite surface, and reduced the adhesion force of water droplet on hematite surface. Scanning Electron Microscope – Energy Dispersive Spectrometer observations confirmed that increasing nano-scaled roughness increased the number of fine quartz particles coating on hematite surface. Combined with the analysis of adhesion of water droplet and observation of fine particles coating on hematite surface, the effect of surface roughness on minerals flotation separation could be better understood.