The invention discloses a preparation method of high-purity mannan oligosaccharide. The preparation method comprises the steps of 1, producing a mannan oligosaccharide mixture through enzymolysis conversion; 2, carrying out centrifugal separation: separating enzymatic hydrolysate through a centrifugal machine; 3, carrying out ceramic membrane filtration: filtering centrifugate through a ceramic membrane group as precision separation to remove protein, starch and polysaccharide substances; 4, carrying out ion exchange refining: desalting, decoloring and deodorizing filtrate through a mannan oligosaccharide ceramic membrane; 5, carrying out 2000 molecular weight ultrafiltration: ultrafiltering ion exchange refining liquid through a polyether sulfone-polyamide composite membrane with interception as Dalton to obtain a polymerization degree; 6, carrying out nanofiltration: filtering sugar liquid through a ceramic membrane, ion exchange refining and an ultrafiltration membrane; and 7, carrying out spray drying: preheating a nano-filtered concentrated solution, and drying in a spray drying tower. The dried product contains more than 95% of mannan oligosaccharide, and has an obvious function of multiplying bifidobacterium; meanwhile, the product, as a most promising substitute, can reduce harmful bacteria.