The invention relates to a preparation method for high-purity D-psicose. The preparation method comprises the following steps: (1) subjecting the fermentation broth of Bacillus subtilis to centrifugation and then subjecting obtained thalli to homogenization so as to obtain a mixed solution containing D-psicose 3-epimerase; (2) preparing a fructose solution, adding the mixed solution containing D-psicose 3-epimerase, adjusting a pH value, carrying out a reaction under a heat insulation condition, then adding the fructose solution into the reaction solution in a fed-batch manner, continuing the reaction, and then stopping the reaction so as to prepare a crude D-psicose solution; and (3) subjecting the crude D-psicose solution to decoloring, filtering, ion exchange, chromatographic separation and concentration, and then to crystallization or drying so as to prepare D-psicose. The preparation method provided by the invention uses Bacillus subtilis fermentation broth for direct production and omits the process of enzyme extraction, so production cost is greatly reduced, a reduction of 25% or so compared with traditional production methods, so the competitiveness of prepared D-psicose is greatly improved.