The invention discloses a preparation method of levoglucosenone. The preparation method comprises the following steps of firstly, dissolving phosphoric acid in water to form a phosphoric acid solution; adding cellulose or cellulose-contained biomass, mixing and soaking; filtering the soaked cellulose or cellulose-contained biomass, pressing and extruding or centrifuging, and performing pyrolysis pretreatment on the treated matter, so as to obtain a dewatered intermediate product; cooling the obtained dewatered intermediate product to room temperature, cooling, putting into a sodium hydroxide or potassium hydroxide water solution, and fully mixing and soaking; filtering the mixed and soaked product, pressing and extruding or centrifuging, performing non-oxygen pyrolysis on the treated product, and collecting condensate in the pyrolysis process, so as to obtain a pyrolysate containing the levoglucosenone. The preparation method of the levoglucosenone has the advantages that by adopting two-step reaction, the yield rate is greatly increased and reaches about 40%; compared with the prior art, the cost is greatly reduced; by adopting the technical scheme, the large application prospect is realized.