Abstract: Reverse thinking is an important way of innovative thinking.In field of modern biochemical separation, its abundant of way of reverse thinking.This paper enumerated and analyzed the way of reverse thinking in the three main procedures, i.e.extraction, primary separation and refining in bio-separation engineering, including positive absorption and negative absorption, fixed bed absorption and expanded bed absorption, two- aqueous phase system, small particle medium and monolithic column, reverse elution, radial chromatography, etc.Finally, this paper gave some suggestion on how to design the teaching.