The invention relates to a preparation method of high fructose corn syrup. The preparation method comprises 1, taking starch as a raw material and orderly carrying out slurry mixing, liquefaction, saccharification, enzyme deactivation, coarse filtration, first ion exchange, first decolorizing filtration, first condensation, isomerization, second ion exchange, second decolorizing filtration and second condensation to obtain a high fructose corn syrup semi-finished product, 2, orderly carrying out chromatographic separation, third ionic exchange, third decolorizing filtration and third condensation on the high fructose corn syrup semi-finished product to obtain a high fructose corn syrup finished product, blending the returned material produced through chromatographic separation and a liquefied liquid to obtain a mixed liquefied liquid, putting the mixed liquefied liquid into a saccharification section, carrying out saccharification and orderly carrying out later processes to obtain a high fructose corn syrup finished product. The preparation method can effectively treat a returned material of a chromatographic separation section, improve returned material tank use efficiency, effectively utilize the returned material in production without influence on product quality, saves raw materials and reduces an enterprise cost.